Integration of a Polynucleotide Encoding a Polypeptide that Catalyzes Pyruvate to Acetolactate Conversion

ABSTRACT

The invention relates to recombinant host cells having at least one integrated polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway, e.g., pyruvate to acetolactate conversion. The invention also relates to methods of increasing the biosynthetic production of isobutanol, 2,3-butanediol, 2-butanol or 2-butanone using such host cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is related to and claims the benefit of priority ofU.S. Provisional Patent Application No. 61/380,563, filed Sep. 7, 2010and U.S. Provisional Patent Application No. 61/466,557, filed Mar. 23,2011, both of which are herein incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: 20110907_CL5178USNA_SeqList.txt, Size: 669,953 bytes,and Date of Creation: Aug. 31, 2011) filed with the application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of industrial microbiology and thefermentative production of butanol and isomers thereof. Morespecifically, the invention relates to recombinant host cells having oneor more integrated polynucleotide encoding a polypeptide that catalyzesa step in a pyruvate-utilizing biosynthetic pathway, e.g., pyruvate toacetolactate conversion.

BACKGROUND OF THE INVENTION

Butanol is an important industrial chemical, useful as a fuel additive,as a feedstock chemical in the plastics industry, and as a foodgradeextractant in the food and flavor industry. Each year 10 to 12 billionpounds of butanol are produced by petrochemical means and the need forthis commodity chemical will likely increase. 2-butanone, also referredto as methyl ethyl ketone (MEK), is a widely used solvent and is themost important commercially produced ketone, after acetone. It is usedas a solvent for paints, resins, and adhesives, as well as a selectiveextractant, activator of oxidative reactions, and can be chemicallyconverted to 2-butanol by reacting with hydrogen in the presence of acatalyst (Nystrom et al., J. Am. Chem. Soc., 69:1198, 1947).2,3-butanediol can be used in the chemical synthesis of butene andbutadiene, important industrial chemicals currently obtained fromcracked petroleum, and esters of 2,3-butanediol can be used asplasticizers (Voloch et al., “Fermentation Derived 2,3-Butanediol,” in:Comprehensive Biotechnology, Pergamon Press Ltd., England, Vol. 2,Section 3, pp. 933-947, 1986).

Microorganisms can be engineered for expression of biosynthetic pathwaysfor the production of products such as 2,3-butanediol, 2-butanone,2-butanol and isobutanol. U.S. Pat. No. 7,851,188 discloses theengineering of recombinant microorganisms for production of isobutanol.U.S. Appl. Pub. Nos. 20070259410 and 20070292927 disclose theengineering of recombinant microorganisms for the production of2-butanone or 2-butanol. Multiple pathways are known for thebiosynthesis of isobutanol and 2-butanol, all of which initiate withcellular pyruvate. Butanediol is an intermediate in the 2-butanolpathway disclosed in U.S. Appl. Pub. No. 20070292927.

Pyruvate metabolism has been altered in yeast for the production oflactic acid and glycerol. U.S. Appl. Pub. No. 20070031950 discloses ayeast strain with a disruption of one or more pyruvate decarboxylase orpyruvate dehydrogenase genes and expression of a D-lactate dehydrogenasegene, which is used for the production of D-lactic acid. Ishida et al.(Biosci. Biotech. and Biochem., 70:1148-1153, 2006) describeSaccharomyces cerevisiae with disrupted pyruvate decarboxylase genes andexpression of lactate dehydrogenase. U.S. Appl. Pub. No. 2005/0059136discloses glucose tolerant C2 carbon source-independent (GCSI) yeaststrains with no pyruvate decarboxylase activity, which can have anexogenous lactate dehydrogenase gene. Nevoigt et al. (Yeast,12:1331-1337, 1996) describe the impact of reduced pyruvatedecarboxylase and increased NAD-dependent glycerol-3-phosphatedehydrogenase in Saccharomyces cerevisiae on glycerol yield.

Stable production of polynucleotides by a yeast cell for pyruvatebiosynthetic pathways are needed for industrial fermentative productionof alcohols or other compounds. Further, there is a need for improvedmeans of isobutanol, 2,3-butanediol, 2-butanol or 2-butanone productionin recombinant host cells such as yeast.

BRIEF SUMMARY OF THE INVENTION

Provided herein are recombinant host cells having one or more integratedpolynucleotides encoding a polypeptide that catalyzes a step in apyruvate-utilizing biosynthetic pathway, e.g., pyruvate to acetolactateconversion. Such host cells provide a means to stabilize and/or increaseproduct formation of a biosynthetic pathway, such as isobutanol,2,3-butanediol, 2-butanol or 2-butanone, compared to host cells which donot have an integrated polynucleotide encoding a polypeptide thatcatalyzes biosynthetic pathway steps such as pyruvate to acetolactateconversion.

One aspect of the invention relates to a recombinant host cellcomprising a polynucleotide encoding a polypeptide which catalyzes theconversion of pyruvate to acetolactate integrated into the chromosome ofthe host cell. In another aspect, the host cell comprises apyruvate-utilizing biosynthetic pathway and a polynucleotide encoding apolypeptide which catalyzes the conversion of pyruvate to acetolactateintegrated into the chromosome of the host cell. In another aspect, thepolynucleotide is heterologous to the host cell.

An aspect of the invention relates to a recombinant host cell comprisingan isobutanol biosynthetic pathway wherein said pathway comprises thesubstrate to product conversion pyruvate to acetolactate catalyzed by apolypeptide encoded by a heterologous polynucleotide integrated into thechromosome and wherein said pathway comprises the substrate to productconversion acetolactate to 2,3-dihydroxyisovalerate catalyzed by apolypeptide encoded by a polynucleotide on a plasmid. In embodiments,the titer of isobutanol production is increased as compared to arecombinant host cell wherein the polynucleotide encoding a polypeptidethat catalyzes the conversion of pyruvate to acetolactate is notintegrated into the chromosome.

An aspect of the invention relates to a recombinant host cell comprisinga 2,3-butanediol, 2-butanol, or 2-butanone biosynthetic pathway whereinsaid pathway comprises the substrate to product conversion pyruvate toacetolactate catalyzed by a polypeptide encoded by a heterologouspolynucleotide integrated into the chromosome and wherein said pathwaycomprises at least one substrate to product conversion catalyzed by apolypeptide encoded by a polynucleotide on a plasmid. In embodiments,the titer of 2,3-butanediol, 2-butanol, or 2-butanone production isincreased as compared to a recombinant host cell wherein thepolynucleotide encoding a polypeptide that catalyzes the conversion ofpyruvate to acetolactate is not integrated into the chromosome.

In another aspect, the invention relates to a recombinant host cellcomprising a first heterologous polynucleotide encoding a firstpolypeptide which catalyzes the conversion of a step of apyruvate-utilizing biosynthetic pathway; a second heterologouspolynucleotide encoding a second polypeptide which catalyzes theconversion of a step of a pyruvate-utilizing biosynthetic pathway; and athird heterologous polynucleotide encoding a third polypeptide whichcatalyzes the conversion of a step of a pyruvate-utilizing biosyntheticpathway; wherein the first and second heterologous polynucleotides areintegrated into the chromosome of the host cell; wherein the thirdheterologous polynucleotide is not integrated into the chromosome of thehost cell; and wherein the first, second, and third polypeptidescatalyze different steps of the pyruvate-utilizing biosynthetic pathway.

In another aspect, the invention relates to a recombinant host cellcomprising (a) a first heterologous polynucleotide encoding a firstpolypeptide which catalyzes a substrate to product conversion ofpyruvate to acetolactate; (b) a second heterologous polynucleotideencoding a second polypeptide which catalyzes the substrate to productconversion of α-ketoisovalerate to isobutyraldehyde; and (c) a thirdheterologous polynucleotide encoding a third polypeptide which catalyzesthe conversion of a step of a isobutanol biosynthetic pathway that isnot the conversion of (a) or (b); wherein the first and secondheterologous polynucleotides are integrated into the chromosome; whereinthe third heterologous polynucleotide is not integrated into thechromosome; and wherein the host cell produces isobutanol.

In another aspect, the invention relates to a recombinant host cellcomprising (a) a first heterologous polynucleotide encoding a firstpolypeptide which catalyzes a substrate to product conversion ofα-ketoisovalerate to isobutyraldehyde; and (b) a second heterologouspolynucleotide encoding a second polypeptide which catalyzes theconversion of a step of a isobutanol biosynthetic pathway that is notthe conversion of (a); wherein the first heterologous polynucleotide isintegrated into the chromosome; wherein the second heterologouspolynucleotide is not integrated into the chromosome; and wherein thehost cell produces isobutanol.

In aspects of the invention, the host cell is a bacterium, acyanobacterium, a filamentous fungus, or a yeast. In another aspect, thehost cell is a member of the genus Clostridium, Zymomonas, Escherichia,Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus,Enterococcus, Alcaligenes, Klebsiella, Issatchenkia, Paenibacillus,Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida,Hansenula, or Saccharomyces. In another aspect, the host cell isEscherichia coli, Alcaligenes eutrophus, Bacillus licheniformis,Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida,Bacillus subtilis, Lactobacillus plantarum, Enterococcus faecium,Enterococcus gallinarium, Enterococcus faecalis or Saccharomycescerevisiae. In another aspect, the host cell is a facultative anaerobe.

In another aspect of the invention, the pyruvate-utilizing biosyntheticpathway comprises one or more polynucleotides encoding polypeptides thatcatalyze a substrate to product conversion of the pathway. In anotheraspect, one or more of the polynucleotides are integrated into thechromosome. In another aspect, the pyruvate-utilizing biosyntheticpathway forms the product 2,3-butanediol, isobutanol, 2-butanol or2-butanone.

In one aspect of the invention, the pyruvate-utilizing biosyntheticpathway is a butanol biosynthetic pathway. In another aspect, thebutanol biosynthetic pathway is a 2-butanol biosynthetic pathway or anisobutanol biosynthetic pathway. In another aspect, the host cellcomprises one or more polynucleotides encoding a polypeptide thatcatalyzes a substrate to product conversion of (i) pyruvate toacetolactate; (ii) acetolactate to 2,3-dihydroxyisovalerate; (iii)2,3-dihydroxyisovalerate to α-ketoisovalerate; (iv) α-ketoisovalerate toisobutyraldehyde; or (v) isobutyraldehyde to isobutanol. In anotheraspect, one or more of the polynucleotides of (ii), (iii), (iv), or (v)are on a plasmid. In another aspect, the host cell comprises one or morepolynucleotides encoding a polypeptide that catalyzes a substrate toproduct conversion of (i) pyruvate to acetolactate; (ii) acetolactate to2,3-dihydroxyisovalerate; (iii) 2,3-dihydroxyisovalerate toα-ketoisovalerate; (iv) α-ketoisovalerate to isobutyryl-CoA; (v)isobutyryl-CoA to isobutyraldehyde; or (vi) isobutyraldehyde toisobutanol. In another aspect, one or more of the polynucleotides of(ii), (iii), (iv), (v), or (vi) are on a plasmid. In another aspect, thehost cell comprises one or more polynucleotides encoding a polypeptidethat catalyzes a substrate to product of (i) pyruvate to acetolactate;(ii) acetolactate to 2,3-dihydroxyisovalerate; (iii)2,3-dihydroxyisovalerate to α-ketoisovalerate; (iv) α-ketoisovalerate tovaline; (v) valine to isobutylamine; (vi) isobutylamine toisobutyraldehyde; or (vii) isobutyraldehyde to isobutanol. In anotheraspect, one or more of the polynucleotides of (ii), (iii), (iv), (v),(vi), or (vii) are on a plasmid.

In another aspect of the invention, the host cell comprises one or morepolynucleotides encoding a polypeptide that catalyzes a substrate toproduct conversion of (i) pyruvate to acetolactate; (ii) acetolactate toacetoin; (iii) acetoin to 2,3-butanediol; or (iv) 2,3-butanediol to2-butanone. In another aspect, one or more of the polynucleotides of(ii), (iii), or (iv) are on a plasmid. In another aspect, the host cellcomprises one or more polynucleotides encoding a polypeptide thatcatalyzes a substrate to product conversion of (i) pyruvate toacetolactate; (ii) acetolactate to acetoin; (iii) acetoin to2,3-butanediol; (iv) 2,3-butanediol to 2-butanone; or (v) 2-butanone to2-butanol. In another aspect, one or more of the polynucleotides of(ii), (iii), (iv), or (v) are on a plasmid.

In another aspect of the invention, the host cell comprises one or morepolynucleotides encoding a polypeptide that catalyzes a substrate toproduct conversion of (i) pyruvate to acetolactate; (ii)alpha-acetolactate to acetoin; (iii) acetoin to 3-amino-2-butanol; (iv)3-amino-2-butanol to 3-amino-2-butanol phosphate; (v) or3-amino-2-butanol phosphate to 2-butanone. In another aspect, one ormore of the polynucleotides of (ii), (iii), (iv), or (v) are on aplasmid. In another aspect, the host cell comprises one or morepolynucleotides encoding a polypeptide that catalyzes a substrate toproduct conversion of (i) pyruvate to acetolactate; (ii)alpha-acetolactate to acetoin; (iii) acetoin to 3-amino-2-butanol; (iv)3-amino-2-butanol to 3-amino-2-butanol phosphate; (v) 3-amino-2-butanolphosphate to 2-butanone.; or (vi) 2-butanone to 2-butanol. In anotheraspect, one or more of the polynucleotides of (ii), (iii), (iv), (v), or(vi) are on a plasmid.

In another aspect, at least one of the polynucleotides encoding apolypeptide that catalyzes a substrate to product conversion isheterologous. In another embodiment, more than one of thepolynucleotides encoding a polypeptide that catalyzes a substrate toproduct conversion are heterologous. In another embodiment, all of thepolynucleotides encoding polypeptides for each of the substrate toproduct conversions of a pyruvate utilizing biosynthetic pathway areheterologous.

In one aspect of the invention, the polypeptide which catalyzes asubstrate to product conversion of pyruvate to acetolactate isacetolactate synthase. In another aspect, the acetolactate synthase hasat least about 80% identity to an amino acid sequence of an acetolactatesynthase described in Table 1. In another aspect, the acetolactatesynthase has at least about 80% identity to an amino acid sequence withSEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, or 18. In another aspect, thepolypeptide which catalyzes the conversion of pyruvate to acetolactatecorresponds to the Enzyme Commission Number EC 2.2.1.6.

In another aspect of the invention, the polypeptide which catalyzes theconversion of acetolactate to 2,3-dihydroxyisovalerate corresponds tothe Enzyme Commission Number EC 1.1.1.86. In another aspect, thepolypeptide which catalyzes the conversion of 2,3-dihydroxyisovalerateto α-ketoisovalerate corresponds to the Enzyme Commission Number EC4.2.1.9. In another aspect, the polypeptide which catalyzes theconversion of α-ketoisovalerate to isobutyraldehyde corresponds to theEnzyme Commission Number EC 4.1.1.72 or 4.1.1.1. In another aspect, thepolypeptide which catalyzes the conversion of isobutyraldehyde toisobutanol corresponds to the Enzyme Commission Number EC 1.1.1.265,1.1.1.1 or 1.1.1.2.

In one aspect, the expression of pyruvate decarboxylase in a host cellof the invention is decreased or substantially eliminated. In anotheraspect, the host cell comprises a deletion, mutation and/or substitutionin an endogenous polynucleotide encoding a polypeptide having pyruvatedecarboxylase activity.

In another aspect of the invention, one or more of the polynucleotidesencoding a polypeptide which catalyzes a step of biosynthetic pathwaydescribed herein are in a plasmid. In another aspect, the plasmidcomprises a sequence at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to one or more of SEQ ID NOs: 1 to 89, or is atleast about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toany one of SEQ ID NOs: 129-133 or a coding region thereof.

In one aspect, the expression of glycerol-3-phosphate dehydrogenase in ahost cell of the invention is decreased or substantially eliminated. Inanother aspect, the expression of FRA2 in a host cell of the inventionis decreased or substantially eliminated. In another aspect, one or moreof the polynucleotides described herein is integrated into thechromosome of the host cell at the PDC1-TRX1 intergenic region.

In one aspect, the invention relates to a method of producing a productof a biosynthetic pathway from a host cell of the invention. In anotheraspect, the invention relates to a method of producing butanol,comprising (a) providing a recombinant host cell of the invention; and(b) contacting the host cell with a fermentable carbon substrate to forma fermentation broth under conditions whereby butanol is produced. Inanother aspect, the method further comprises contacting the fermentationbroth with an extractant to produce a two-phase fermentation mixture. Inanother aspect, the extractant comprises fatty acids. In another aspect,the fatty acids are derived from corn oil or soybean oil. In anotheraspect, the extractant comprises a water immiscible organic extractantsuch as C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂to C₂₂ fatty acids, C₁₂ to C₂₂ fatty amides, or C₁₂ to C₂₂ fattyaldehydes. In another aspect, the method further comprises contactingthe fermentation broth with an organic acid and an enzyme capable ofesterifying the butanol with the organic acid. In another aspect, themethod further comprises vaporizing at least a portion of thefermentation broth to form a vapor stream comprising water and butanol.

In one aspect, the rate of butanol production from a host cell of theinvention is increased by at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 2-fold, at least about 3-fold, or atleast about 4-fold greater as compared to a host cell that does not havea polynucleotide encoding a polypeptide that catalyzes the conversion ofpyruvate to acetolactate integrated into the chromosome. In anotheraspect, the titer of butanol production from a host cell of theinvention is increased by at least about 10%, at least about 20%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 2-fold, at least about 3-fold, or atleast about 4-fold greater as compared to a host cell that does not havea polynucleotide encoding a polypeptide that catalyzes the conversion ofpyruvate to acetolactate integrated into the chromosome.

In another aspect, the invention relates to a method for increasing thecopy number or expression of a non-integrated recombinant polynucleotideencoding a polypeptide that catalyzes a step of a biosynthetic pathwaydescribed herein, comprising contacting a host cell of the inventionwith a fermentable carbon substrate to form a fermentation broth underconditions whereby the product of the biosynthetic pathway is produced.In another aspect, the invention relates to a method for increasing theflux in a pyruvate-utilizing biosynthetic pathway comprising: (a)providing a recombinant host cell of the invention; and (b) contactingthe host cell with a fermentable carbon substrate to form a fermentationbroth under conditions whereby the flux in the pyruvate-utilizingbiosynthetic pathway in the host cell is increased.

In another aspect, the invention relates to a method of producing arecombinant host cell comprising transforming the host cell with (i) oneor more polynucleotides encoding a polypeptide that catalyzes asubstrate to product conversion of a pyruvate-utilizing biosyntheticpathway; and (ii) a polynucleotide encoding a peptide that catalyzes theconversion of pyruvate to acetolactate; wherein the polynucleotide of(ii) is integrated into the chromosome. In another aspect, the inventionrelates to a method of increasing the formation of a product of apyruvate-utilizing biosynthetic pathway comprising (i) providing arecombinant host cell of the invention; and (ii) growing the host cellunder conditions wherein the product of the pyruvate-utilizing pathwayis formed at an amount of product greater than the amount of productformed by a host cell comprising a polynucleotide encoding a polypeptidewhich catalyzes the conversion of pyruvate to acetolactate that is notintegrated into the chromosome.

In another aspect, the invention relates to a composition comprising (i)a host cell of the invention; (ii) butanol; and (iii) an extractant. Inanother aspect, the invention relates to a composition comprising (i) ahost cell of the invention; (ii) butanol; (iii) an extractant; and (iv)an esterification enzyme. In another aspect, the butanol of suchcomposition is isobutanol.

In another aspect, the invention relates to a method for chromosomallyintegrating acetolactate synthase (als) into a yeast host cellcomprising transforming said host cell with an integration vectorcomprising SEQ ID NO: 131. In another aspect, the host cell furthercomprises an isobutanol biosynthetic pathway. In another aspect, thehost comprises at least two chromosomally integrated polynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate the present invention and, togetherwith the description, further serve to explain the principles of theinvention and to enable a person skilled in the pertinent art to makeand use the invention.

FIG. 1 shows pathways and enzymes for pyruvate utilization.

FIG. 2 shows three different isobutanol biosynthetic pathways

FIG. 3 shows four different 2-butanol biosynthetic pathways.

FIGS. 4A-4D show sequence relationships of acetolactate synthase (als)coding regions that were retrieved by BLAST analysis using the sequenceof B. subtilis AlsS, limiting to the 100 closest neighbors. The alsencoding sequence is identified by its source organism.

FIG. 5 shows the PNY2204 locus (pdc1Δ::ilvD::pUC19-kan::FBA-alsS::TRX1).

FIG. 6 shows the PNY2211 locus (pdc1Δ::ilvD::FBA-alsS::TRX1). The alsSgene integration in the pdc1-trx1 intergenic region is considered a“scarless” insertion since vector, marker gene and loxP sequences arelost.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions which form a partof this application.

The following sequences conform with 37 C.F.R. §§1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (2009) and the sequence listing requirements of the EPO and PCT[Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions]. The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

TABLE 1 SEQ ID Numbers of Coding Regions and Proteins Referred to HereinSEQ ID NO: SEQ ID NO: Description Nucleic acid Amino acid Klebsiellapneumoniae budB (acetolactate synthase) 1  2 Bacillus subtilis alsS 3  4(acetolactate synthase) Lactococcus lactis als 5  6 (acetolactatesynthase) Als Staphylococcus aureus 7  8 Als Listeria monocytogenes 9 10Als Streptococcus mutans 11 12 Als Streptococcus thermophiles 13 14 AlsVibrio angustum 15 16 Als Bacillus cereus 17 18 budA, acetolactatedecarboxylase from Klebsiella 19 20 pneumoniae ATCC 25955 alsD,acetolactate decarboxylase from Bacillus subtilis 21 22 budA,acetolactate decarboxylase from Klebsiella terrigena 23 24 budC,butanediol dehydrogenase from Klebsiella 25 26 pneumoniae IAM1063butanediol dehydrogenase from Bacillus cereus 27 28 butB, butanedioldehydrogenase from Lactococcus lactis 29 30 RdhtA, B12-indep dioldehydratase from Roseburia 31 32 inulinivorans RdhtB, B12-indep dioldehydratase reactivase from 33 34 Roseburia inulinivorans sadB, butanoldehydrogenase from Achromobacter 35 36 xylosoxidans S. cerevisiae ILV537 38 (acetohydroxy acid reductoisomerase) Vibrio cholerae ketol-acidreductoisomerase 39 40 Pseudomonas aeruginosa ketol-acidreductoisomerase 41 42 Pseudomonas fluorescens ketol-acidreductoisomerase 43 44 S. cerevisiae ILV3 45 46 (Dihydroxyaciddehydratase; DHAD) Lactococcus lactis kivD (branched-chain α-keto acid47 48 decarboxylase), codon optimized Lactococcus lactis kivD(branched-chain α-keto acid 49  48* decarboxylase) Pf5.IlvC-Z4B8 mutantPseudomonas fluorescens 82 83 acetohydroxy acid reductoisomeraseBacillis subtilis kivD codon optimized for S. cerevisiae 84 85expression Equus caballus alcohol dehydrogenase codon optimized for 8687 S. cerevisiae expression Streptococcus mutans ilvD (DHAD) 88 89 K9G9variant of Anaerostipes caccae KARI — 225  K9D3 variant of Anaerostipescaccae KARI — 224  Beijerinkia indica ADH — 237 Ketoisovaleratedecarboxylase from Listeria grayi — 247  Ketoisovalerate decarboxylasefrom Macrococcus — 248  caseolyticus *The same amino acid sequence isencoded by SEQ ID NOs: 47 and 49.

TABLE 2 SEQ ID Numbers of Target Gene Coding Regions and ProteinsReferred to Herein SEQ ID NO: SEQ ID NO: Description Nucleic acid Aminoacid PDC1 pyruvate decarboxylase from 50 51 Saccharomyces cerevisiaePDC5 pyruvate decarboxylase from 52 53 Saccharomyces cerevisiae PDC6pyruvate decarboxylase from 54 55 Saccharomyces cerevisiae pyruvatedecarboxylase from Candida 56 57 glabrata PDC1 pyruvate decarboxylasefrom Pichia 58 59 stipites PDC2 pyruvate decarboxylase from Pichia 60 61stipites pyruvate decarboxylase from Kluyveromyces 62 63 lactis pyruvatedecarboxylase from Yarrowia 64 65 lipolytica pyruvate decarboxylase from66 67 Schizosaccharomyces pombe GPD1 NAD-dependent glycerol-3-phosphate68 69 dehydrogenase from Saccharomyces cerevisiae GPD2 NAD-dependentglycerol-3-phosphate 70 71 dehydrogenase from Saccharomyces cerevisiaeGPD1 NAD-dependent glycerol-3-phosphate 72 73 dehydrogenase from Pichiastipitis GPD2 NAD-dependent glycerol-3-phosphate 74 75 dehydrogenasefrom Pichia stipites NAD-dependent glycerol-3-phosphate 76 77dehydrogenase from Kluyveromyces thermotolerans GPD1 NAD-dependentglycerol-3-phosphate 78 79 dehydrogenase from Schizosaccharomyces pombeGPD2 NAD-dependent glycerol-3-phosphate 80 81 dehydrogenase fromSchizosaccharomyces pombe AFT1 from Saccharomyces cerevisiae 227 228AFT2 from Saccharomyces cerevisiae 229 230 FRA2 from Saccharomycescerevisiae 231 232 GRX3 from Saccharomyces cerevisiae 233 234 CCC1 fromSaccharomyces cerevisiae 235 236 ALD6 from Saccharomyces cerevisiae —223 YMR226C from Saccharomyces cerevisiae — 226

SEQ ID NOs:90-222 and 243-246 are sequences used and described in theExamples.

SEQ ID NOs: 238-242 are hybrid promoter sequences referred to herein.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference, unless only specific sections of patents orpatent publications are indicated to be incorporated by reference.

The materials, methods and examples are illustrative only and are notintended to be limiting. Other features and advantages of the inventionwill be apparent from the detailed description and from the claims.

The present invention relates to recombinant microorganisms and methodsfor the production of butanol. The present invention meets a number ofcommercial and industrial needs. Butanol is an important industrialcommodity chemical with a variety of applications, where its potentialas a fuel or fuel additive is particularly significant. Although only afour-carbon alcohol, butanol has energy content similar to that ofgasoline and can be blended with any fossil fuel. Butanol is favored asa fuel or fuel additive as it yields only CO₂ and little or no SO_(X) orNO_(X) when burned in the standard internal combustion engine.Additionally butanol is less corrosive than ethanol, another fueladditive.

In addition to its utility as a biofuel or fuel additive, butanol hasthe potential of impacting hydrogen distribution problems in theemerging fuel cell industry. Fuel cells today are plagued by safetyconcerns associated with hydrogen transport and distribution. Butanolcan be easily reformed for its hydrogen content and can be distributedthrough existing gas stations in the purity required for either fuelcells or vehicles.

The following definitions and abbreviations are to be used for theinterpretation of the claims and the specification.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains,” or “containing,” or any othervariation thereof, are intended to be non-exclusive or open-ended. Forexample, a composition, a mixture, a process, a method, an article, oran apparatus that comprises a list of elements is not necessarilylimited to only those elements but may include other elements notexpressly listed or inherent to such composition, mixture, process,method, article, or apparatus. Further, unless expressly stated to thecontrary, “or” refers to an inclusive or and not to an exclusive or. Forexample, a condition A or B is satisfied by any one of the following: Ais true (or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

As used herein, the term “consisting essentially of” in the context of aclaim is intended to represent the intermediate ground between a closedclaim written in a “consisting of” format and a fully open claim writtenin a “comprising” format. See M.P.E.P. §2111.03.

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances, i.e., occurrences of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or to carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about,” the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, preferably within 5% of the reported numerical value.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the specification and the claims.

The term “butanol” as used herein, refers to 2-butanol, isobutanol ormixtures thereof.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathwayto produce isobutanol from pyruvate.

The term “2-butanol biosynthetic pathway” refers to an enzyme pathway toproduce 2-butanol from pyruvate.

The term “2-butanone biosynthetic pathway” refers to an enzyme pathwayto produce 2-butanone from pyruvate.

The term “extractant” as used herein refers to one or more organicsolvents which are used to extract butanol and/or other components froma fermentation broth.

The terms “acetolactate synthase” and “acetolactate synthetase” are usedinterchangeably herein to refer to an enzyme that catalyzes theconversion of pyruvate to acetolactate and CO₂. Examples of acetolactatesynthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992,Academic Press, San Diego). These enzymes are available from a number ofsources, including, but not limited to, Bacillus subtilis [GenBank Nos:CAB15618 and Z99122, NCBI (National Center for BiotechnologyInformation) amino acid sequence, NCBI nucleotide sequence,respectively], Klebsiella pneumoniae (GenBank Nos: AAA25079 and M73842),and Lactococcus lactis (GenBank Nos: AAA25161 and L16975). Additionalexamples are also provided in Table 1.

The term “acetolactate decarboxylase” refers to a polypeptide (orpolypeptides) having an enzyme activity that catalyzes the conversion ofalpha-acetolactate to acetoin. Examples of acetolactate decarboxylasesare known as EC 4.1.1.5 and are available, for example, from Bacillussubtilis (DNA: SEQ ID NO:21, Protein: SEQ ID NO:22), Klebsiellaterrigena (DNA: SEQ ID NO:23, Protein: SEQ ID NO:24) and Klebsiellapneumoniae (DNA: SEQ ID NO:19, protein: SEQ ID NO:20).

The term “acetoin aminase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of acetoin to3-amino-2-butanol. Acetoin aminase may utilize the cofactor pyridoxal5′-phosphate or NADH (reduced nicotinamide adenine dinucleotide) orNADPH (reduced nicotinamide adenine dinucleotide phosphate). Theresulting product may have (R) or (S) stereochemistry at the 3-position.The pyridoxal phosphate-dependent enzyme may use an amino acid such asalanine or glutamate as the amino donor. The NADH- and NADPH-dependentenzymes may use ammonia as a second substrate. A suitable example of anNADH-dependent acetoin aminase, also known as amino alcoholdehydrogenase, is described by Ito et al. (U.S. Pat. No. 6,432,688). Anexample of a pyridoxal-dependent acetoin aminase is the amine:pyruvateaminotransferase (also called amine:pyruvate transaminase) described byShin and Kim (J. Org. Chem. 67:2848-2853 (2002)).

The term “aminobutanol kinase” refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of3-amino-2-butanol to 3-amino-2-butanol O-phosphate. Aminobutanol kinasemay utilize ATP as the phosphate donor. There are reports of enzymesthat catalyze the analogous reaction on the similar substratesethanolamine and 1-amino-2-propanol (Jones et al. (1973) Biochem. J.134:167-182). U.S. Appl. Pub. No. 20070292927 describes, in Example 14,an amino alcohol kinase of Erwinia carotovora subsp. atroseptica.

The term “aminobutanol phosphate phospho-lyase”, also called “aminoalcohol 0-phosphate lyase”, refers to a polypeptide (or polypeptides)having an enzyme activity that catalyzes the conversion of3-amino-2-butanol O-phosphate to 2-butanone. Aminobutanol phosphatephospho-lyase may utilize the cofactor pyridoxal 5′-phosphate. There arereports of enzymes that catalyze the analogous reaction on the similarsubstrate 1-amino-2-propanol phosphate (Jones et al. (1973) Biochem J.134:167-182). U.S. Appl. Pub. No. 20070292927 describes, in Example 15,a newly identified aminobutanol phosphate phospho-lyase from theorganism Erwinia carotovora.

The term “butanediol dehydrogenase” also known as “acetoin reductase”refers to a polypeptide (or polypeptides) having an enzyme activity thatcatalyzes the conversion of acetoin to 2,3-butanediol. Butanedioldehydrogenases are a subset of the broad family of alcoholdehydrogenases. Butanediol dehydrogenase enzymes can have specificityfor production of (R)- or (S)-stereochemistry in the alcohol product.Examples of (S)-specific butanediol dehydrogenases are known as EC1.1.1.76 and are available, for example, from Klebsiella pneumoniae(DNA: SEQ ID NO:25, protein: SEQ ID NO:26). Examples of (R)-specificbutanediol dehydrogenases are known as EC 1.1.1.4 and are available, forexample, from Bacillus cereus (DNA: SEQ ID NO:27, protein: SEQ IDNO:28), and Lactococcus lactis (DNA: SEQ ID NO:29, protein: SEQ IDNO:30).

The terms “acetohydroxy acid isomeroreductase” and “acetohydroxy acidreductoisomerase” and “ketol-acid reductoisomerase” (KARI) are usedinterchangeably herein to refer to an enzyme that catalyzes theconversion of acetolactate to 2,3-dihydroxyisovalerate. Suitable enzymesutilize NADH (reduced nicotinamide adenine dinucleotide) and/or NADPH aselectron donor. Examples of acetohydroxy acid isomeroreductases areknown by the EC number 1.1.1.86 and sequences are available from a vastarray of microorganisms, including, but not limited to, Escherichia coli(GenBank Nos: NP_418222 and NC_000913), Saccharomyces cerevisiae(GenBank Nos: NP_013459 and NC_001144), Methanococcus maripaludis(GenBank Nos: CAF30210 and BX957220), and Bacillus subtilis (GenBankNos: CAB14789 and Z99118).

The term “acetohydroxy acid dehydratase” refers to an enzyme thatcatalyzes the conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate. Examples of acetohydroxy acid dehydratases are knownby the EC number 4.2.1.9. These enzymes are available from a vast arrayof microorganisms, including, but not limited to, E. coli (GenBank Nos:YP_026248 and NC_000913), S. cerevisiae (GenBank Nos: NP_012550 andNC_001142), M. maripaludis (GenBank Nos: CAF29874 and BX957219), and B.subtilis (GenBank Nos: CAB14105 and Z99115).

The term “branched-chain α-keto acid decarboxylase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyraldehydeand CO₂. Examples of branched-chain α-keto acid decarboxylases are knownby the EC number 4.1.1.72 and are available from a number of sources,including, but not limited to, Lactococcus lactis (GenBank Nos:AAS49166, AY548760, CAG34226, and AJ746364), Salmonella typhimurium(GenBank Nos: NP_461346 and NC_003197), and Clostridium acetobutylicum(GenBank Nos: NP_149189 and NC_001988).

The term “branched-chain alcohol dehydrogenase” refers to an enzyme thatcatalyzes the conversion of isobutyraldehyde to isobutanol. Examples ofbranched-chain alcohol dehydrogenases are known by the EC number1.1.1.265, but may also be classified under other alcohol dehydrogenases(specifically, EC 1.1.1.1 or 1.1.1.2). These enzymes utilize NADH(reduced nicotinamide adenine dinucleotide) and/or NADPH as electrondonor and are available from a number of sources, including, but notlimited to, S. cerevisiae (GenBank Nos: NP_010656, NC_001136; NP_014051;and NC_001145), E. coli (GenBank Nos: NP_417484 and NC_000913) and C.acetobutylicum (GenBank Nos: NP_349892, NC_003030; NP_349891, andNC_003030).

The term “branched-chain keto acid dehydrogenase” refers to an enzymethat catalyzes the conversion of α-ketoisovalerate to isobutyryl-CoA(isobutyryl-coenzyme A), using NAD⁺ (nicotinamide adenine dinucleotide)as electron acceptor. Examples of branched-chain keto aciddehydrogenases are known by the EC number 1.2.4.4. These branched-chainketo acid dehydrogenases are comprised of four subunits and sequencesfrom all subunits are available from a vast array of microorganisms,including, but not limited to, B. subtilis (GenBank Nos: CAB14336,Z99116, CAB14335, Z99116, CAB14334, Z99116, CAB14337, and Z99116) andPseudomonas putida (GenBank Nos: AAA65614, M57613, AAA65615, M57613,AAA65617, M57613, AAA65618, and M57613).

The term “acylating aldehyde dehydrogenase” refers to an enzyme thatcatalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, usingeither NADH or NADPH as electron donor. Examples of acylating aldehydedehydrogenases are known by the EC numbers 1.2.1.10 and 1.2.1.57. Theseenzymes are available from multiple sources, including, but not limitedto, Clostridium beijerinckii (GenBank Nos: AAD31841 and AF157306), C.acetobutylicum (GenBank Nos: NP_149325, NC_001988, NP_149199, andNC_001988), P. putida (GenBank Nos: AAA89106 and U13232), and Thermsthermophilus (GenBank Nos: YP_145486 and NC_006461).

The term “transaminase” refers to an enzyme that catalyzes theconversion of α-ketoisovalerate to L-valine, using either alanine orglutamate as amine donor. Examples of transaminases are known by the ECnumbers 2.6.1.42 and 2.6.1.66. These enzymes are available from a numberof sources. Examples of sources for alanine-dependent enzymes include,but are not limited to, E. coli (GenBank Nos: YP_026231 and NC_000913)and Bacillus licheniformis (GenBank Nos: YP_093743 and NC_006322).Examples of sources for glutamate-dependent enzymes include, but are notlimited to, E. coli (GenBank Nos: YP_026247 and NC_000913), S.cerevisiae (GenBank Nos: NP_012682 and NC_001142) and Methanobacteriumthermoautotrophicum (GenBank Nos: NP_276546 and NC_000916).

The term “valine dehydrogenase” refers to an enzyme that catalyzes theconversion of α-ketoisovalerate to L-valine, using NAD(P)H as electrondonor and ammonia as amine donor. Examples of valine dehydrogenases areknown by the EC numbers 1.4.1.8 and 1.4.1.9 and are available from anumber of sources, including, but not limited to, Streptomycescoelicolor (GenBank Nos: NP_628270 and NC_003888) and B. subtilis(GenBank Nos: CAB14339 and Z99116).

The term “valine decarboxylase” refers to an enzyme that catalyzes theconversion of L-valine to isobutylamine and CO₂. Examples of valinedecarboxylases are known by the EC number 4.1.1.14. These enzymes arefound in Streptomycetes, such as for example, Streptomyces viridifaciens(GenBank Nos: AAN10242 and AY116644).

The term “omega transaminase” refers to an enzyme that catalyzes theconversion of isobutylamine to isobutyraldehyde using a suitable aminoacid as amine donor. Examples of omega transaminases are known by the ECnumber 2.6.1.18 and are available from a number of sources, including,but not limited to, Alcaligenes denitrificans (GenBank Nos: AAP92672 andAY330220), Ralstonia eutropha (GenBank Nos: YP_294474 and NC_0073479),Shewanella oneidensis (GenBank Nos: NP_719046 and NC_004347), and P.putida (GenBank Nos: AAN66223 and AE016776).

The term “isobutyryl-CoA mutase” refers to an enzyme that catalyzes theconversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzymeB₁₂ as cofactor. Examples of isobutyryl-CoA mutases are known by the ECnumber 5.4.99.13. These enzymes are found in a number of Streptomycetes,including, but not limited to, Streptomyces cinnamonensis (GenBank Nos:AAC08713, U67612, CAB59633, and AJ246005), S. coelicolor (GenBank Nos:CAB70645, AL939123, CAB92663, and L939121), and Streptomyces avermitilis(GenBank Nos: NP_824008, NC_003155, NP_824637 and NC_003155).

The term “substantially free” when used in reference to the presence orabsence of enzyme activities (e.g., pyruvate decarboxylase) in carbonpathways that compete with the present isobutanol pathway means that thelevel of the enzyme is substantially less than that of the same enzymein the wildtype host, where less than about 20% of the wildtype level ispreferred and less than about 15% or 10% of the wildtype level are morepreferred. The activity can be less than about 5%, 4%, 3%, 2% or 1% ofwildtype activity.

The term “a facultative anaerobe” refers to a microorganism that cangrow in both aerobic and anaerobic environments.

The term “carbon substrate” or “fermentable carbon substrate” refers toa carbon source capable of being metabolized by host organisms of thepresent invention and particularly carbon sources selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,and one-carbon substrates or mixtures thereof. Sources for carbonsubstrates can include any feedstock, such as renewable-sourcefeedstocks, including but not limited to any sugar or starch containingbiomass such as corn, wheat, sugar cane, wood, algae; any agriculturalwastes or residues and any lignocellulosic and/or hemicellulosicmaterials.

The term “gene” refers to a nucleic acid fragment that is capable ofbeing expressed as a specific protein, optionally including regulatorysequences preceding (5′ non-coding sequences) and following (3′non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene can comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign gene” or “heterologous gene” refers to a genenot normally found in the host organism, but that is introduced into thehost organism, e.g. by gene transfer, or is found or is native to a hostorganism but is modified in some way to affect its functioning. Apolynucleotide integrated (whether a nature or non-nativepolynucleotide) into a chromosome as described herein is considered aheterologous polynucleotide. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

As used herein the terms “coding sequence” and “coding region” refer toa DNA sequence that codes for a specific amino acid sequence. “Suitableregulatory sequences” refer to nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences can include promoters, translation leadersequences, introns, polyadenylation recognition sequences, RNAprocessing site, effector binding site and stem-loop structure.

The term “promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters can be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters.” It isfurther recognized that because in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of effecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression” as used herein refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression can also refer totranslation of mRNA into a polypeptide.

As used herein the term “transformation” refers to the transfer of anucleic acid fragment into a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements can be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

As used herein, the term “codon degeneracy” refers to the nature of thegenetic code permitting variation of the nucleotide sequence withoutaffecting the amino acid sequence of an encoded polypeptide. The skilledartisan is well aware of the “codon-bias” exhibited by a specific hostcell in usage of nucleotide codons to specify a given amino acid.Therefore, when synthesizing a gene for improved expression in a hostcell, it is desirable to design the gene such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “endogenous” as used herein refers to something that isproduced or synthesized by the organism or that is added to thesurroundings of the organism.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA. Such optimizationincludes replacing at least one, or more than one, or a significantnumber, of codons with one or more codons that are more frequently usedin the genes of that organism.

As used herein, an “isolated nucleic acid fragment” or “isolated nucleicacid molecule” are used interchangeably and mean a polymer of RNA or DNAthat is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. An isolated nucleic acidfragment in the form of a polymer of DNA can be comprised of one or moresegments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid fragment is “hybridizable” to another nucleic acidfragment, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid fragment can anneal to theother nucleic acid fragment under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of conditions uses a series of washesstarting with 6×SSC, 0.5% SDS at room temperature for 15 min, thenrepeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeatedtwice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. Another set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another set ofhighly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDSat 65° C. An additional set of stringent conditions includehybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1%SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. In another embodiment, a minimum length for a hybridizablenucleic acid is at least about 15 nucleotides, at least about 20nucleotides, or at least about 30 nucleotides. Furthermore, the skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such aslength of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Altschul et al., J. Mol.Biol., 215:403-410 (1993)). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides can be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases can be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches the complete amino acidand nucleotide sequence encoding particular fungal proteins. The skilledartisan, having the benefit of the sequences as reported herein, may nowuse all or a substantial portion of the disclosed sequences for purposesknown to those skilled in this art. Accordingly, the instant inventioncomprises the complete sequences as reported in the accompanyingSequence Listing, as well as substantial portions of those sequences asdefined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

The term “percent identity,” as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: (1) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); (2)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); (3) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); (4)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and (5) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the bestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Sequence alignments and percent identity calculations may be performedusing the MegAlign™ program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequencesis performed using the “Clustal method of alignment”which encompassesseveral varieties of the algorithm including the “Clustal V method ofalignment” corresponding to the alignment method labeled Clustal V(described by Higgins et al., CABIOS. 5:151-153, 1989; Higgins et al.,Comput. Appl. Biosci., 8:189-191, 1992) and found in the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.).For multiple alignments, the default values correspond to GAP PENALTY=10and GAP LENGTH PENALTY=10. Default parameters for pairwise alignmentsand calculation of percent identity of protein sequences using theClustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5,WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences usingthe Clustal V program, it is possible to obtain a “percent identity” byviewing the “sequence distances” table in the same program. Additionallythe “Clustal W method of alignment” is available and corresponds to thealignment method labeled Clustal W (described by Higgins et al., CABIOS.5:151-153 (1989); Higgins et al., Comput. Appl. Biosci. 8:189-191(1992))and found in the MegAlign™ v6.1 program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc.). Default parameters for multiplealignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series,DNA Weight Matrix=IUB). After alignment of the sequences using theClustal W program, it is possible to obtain a “percent identity” byviewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to: 24%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95%, or any integer percentage from 24% to 100% may beuseful in describing the present invention, such as 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99%. Suitable nucleic acid fragments not only have the above homologiesbut typically encode a polypeptide having at least 50 amino acids, atleast 100 amino acids, at least 150 amino acids, at least 200 aminoacids, or at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” can be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: (1) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); (2) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.,215:403-410 (1990)); (3) DNASTAR (DNASTAR, Inc. Madison, Wis.); (4)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and (5) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook, J., Fritsch, E. F.and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987). Additional methods arein Methods in Enzymology, Volume 194, Guide to Yeast Genetics andMolecular and Cell Biology (Part A, 2004, Christine Guthrie and GeraldR. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Othermolecular tools and techniques are known in the art and include splicingby overlapping extension polymerase chain reaction (PCR) (Yu, et al.(2004) Fungal Genet. Biol. 41:973-981), positive selection for mutationsat the URA3 locus of Saccharomyces cerevisiae (Boeke, J. D. et al.(1984) Mol. Gen. Genet. 197, 345-346; M A Romanos, et al. Nucleic AcidsRes. 1991 Jan. 11; 19(1): 187), the cre-lox site-specific recombinationsystem as well as mutant lox sites and FLP substrate mutations (Sauer,B. (1987) Mol Cell Biol 7: 2087-2096; Senecoff, et al. (1988) Journal ofMolecular Biology, Volume 201, Issue 2, Pages 405-421; Albert, et al.(1995) The Plant Journal. Volume 7, Issue 4, pages 649-659), “seamless”gene deletion (Akada, et al. (2006) Yeast; 23(5):399-405), and gaprepair methodology (Ma et al., Genetics 58:201-216; 1981).

Biosynthetic Pathway Production Through Conversion of Pyruvate toAcetolactate

Microbial cells produce pyruvate from sugars, which is then utilized ina number of pathways of cellular metabolism including those shown inFIG. 1. Microbial host cells can be engineered to produce a number ofdesirable products with the initial biosynthetic pathway step beingconversion of endogenous pyruvate to acetolactate. Engineeredbiosynthetic pathways for synthesis of isobutanol (FIG 2) are describedin U.S. Appl. Pub. No. 20070092957, which is herein incorporated byreference, and for synthesis of 2-butanol and 2-butanone (FIG 3) aredescribed in U.S. Appl. Pub. Nos. 20070259410 and 20070292927, which areherein incorporated by reference. The product 2,3-butanediol is anintermediate in the biosynthetic pathway described in U.S. Appl. Pub.No. 20070292927. Each of these pathways has the initial step ofconverting pyruvate to acetolactate by acetolactate synthase. Therefore,product yield from these biosynthetic pathways will in part depend uponthe amount of acetolactate that can be produced from pyruvate and theamount of pyruvate that is available.

Applicants have discovered that a recombinant host cell comprising apolynucleotide encoding a polypeptide which catalyzes the conversion ofpyruvate to acetolactate integrated into the chromosome of the host cellcan have improved production of a product of a pyruvate-utilizingbiosynthetic pathway (e.g., a butanol such as isobutanol). Applicantsfound that host cells of the invention can have improved butanolproduction as shown by increased product titer, increased productionrate or increased cell density compared to cells wherein thepolynucleotide is not integrated into the chromosome.

In embodiments, the present invention relates to a recombinant host cellcomprising a pyruvate-utilizing biosynthetic pathway and apolynucleotide encoding a polypeptide which catalyzes the conversion ofpyruvate to acetolactate integrated into the chromosome of the hostcell. In embodiments, the polynucleotide is heterologous to the hostcell. In embodiments, the pyruvate-utilizing biosynthetic pathwaycomprises one or more polynucleotides encoding a polypeptide thatcatalyzes substrate to product conversions of the pathway. Inembodiments, one or more of the polynucleotides are integrated into thechromosome of the host cell.

Expression and Integration of Acetolactate Synthase

In embodiments of the invention, a polypeptide that catalyzes asubstrate to product conversion of pyruvate to acetolactate is anacetolactate synthase. Endogenous acetolactate synthase in a host cellof the invention can be encoded in the mitochondrial genome andexpressed in the mitochondria. In embodiments, to prepare a recombinanthost cell of the present invention (such as yeast), a geneticmodification is made that provides cytosolic expression of acetolactatesynthase. In such embodiments, acetolactate synthase is expressed fromthe nucleus and no mitochondrial targeting signal is included so thatthe enzyme remains in the cytosol (cytosol-localized). Cytosolicexpression of acetolactate synthase is described in US ApplicationPublication No. 20090305363.

Acetolactate synthase enzymes, which also can be called acetohydroxyacid synthase, belong to EC 2.2.1.6 (switched from 4.1.3.18 in 2002),and are well-known. These enzymes participate in the biosyntheticpathway for the proteinogenic amino acids leucine and valine, as well asin the pathway for fermentative production of 2,3-butanediol and acetoinin a number of organisms.

The skilled person will appreciate that polypeptides having acetolactatesynthase activity isolated from a variety of sources can be useful inthe present invention independent of sequence homology. Suitableacetolactate synthase enzymes are available from a number of sources, asdescribed in the definitions. Acetolactate synthase enzyme activitiesthat have substrate preference for pyruvate over ketobutyrate are ofparticular utility, such as those encoded by alsS of Bacillus and budBof Klebsiella (Gollop et al., J. Bacteriol. 172(6):3444-3449, 1990; andHoltzclaw et al., J. Bacteriol. 121(3):917-922, 1975).

Because acetolactate synthases are well known, and because of theprevalence of genomic sequencing, suitable acetolactate synthases can bereadily identified by one skilled in the art on the basis of sequencesimilarity using bioinformatics approaches. Typically BLAST (describedabove) searching of publicly available databases with known acetolactatesynthase amino acid sequences, such as those provided herein, is used toidentify acetolactate synthases, and their encoding sequences, that maybe used in the present strains. For example, acetolactate synthases thatare the 100 closest neighbors of the B. subtilis AlsS sequence aredepicted in a phylogenetic tree in FIG. 4. The homology relationshipsbetween the sequences identified are shown in this tree. Among thesesequences are those having 40% identity, yet these have been verified asacetolactate synthases. Acetolactate synthase proteins having at leastabout 70-75%, 75%-80%, 80-85%, 85%-90%, 90%-95% or at least about 98% or99% sequence identity to any of the acetolactate synthase proteins inTable 1, or any of the acetolactate synthase proteins represented inFIG. 4 can be used in the present strains. Identities are based on theClustal W method of alignment using the default parameters of GAPPENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of proteinweight matrix.

Examples of sequences encoding acetolactate synthase which can be usedto provide cytosolic expression of acetolactate synthase (als) activityare listed in Table 1. Additional acetolactate synthase encodingsequences that can be used for yeast cytosolic expression can beidentified in the literature and in bioinformatics databases well knownto the skilled person, and some coding regions for als proteins arerepresented in FIG. 4 by the source organism. Any als having EC number2.2.1.6 can be identified by one skilled in the art and can be used inthe present host cells.

Additionally, the sequences described herein or those recited in the artcan be used to identify other homologs in nature. For example, each ofthe acetolactate synthase encoding nucleic acid fragments describedherein may be used to isolate genes encoding homologous proteins.Isolation of homologous genes using sequence-dependent protocols is wellknown in the art. Examples of sequence-dependent protocols include, butare not limited to, (1) methods of nucleic acid hybridization; (2)methods of DNA and RNA amplification, as exemplified by various uses ofnucleic acid amplification technologies [e.g., polymerase chain reaction(PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction(LCR), Tabor et al., Proc. Acad. Sci. USA 82:1074, 1985; or stranddisplacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci.USA., 89:392, 1992]; and (3) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to theacetolactate synthase encoding genes described herein can be isolateddirectly by using all or a portion of the instant nucleic acid fragmentsas DNA hybridization probes to screen libraries from any desiredorganism using methodology well known to those skilled in the art.Specific oligonucleotide probes based upon the disclosed nucleic acidsequences can be designed and synthesized by methods known in the art(Maniatis, supra). Moreover, the entire sequences can be used directlyto synthesize DNA probes by methods known to the skilled artisan (e.g.,random primers DNA labeling, nick translation or end-labelingtechniques), or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of (or full-length of) the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length DNA fragments by hybridizationunder conditions of appropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein et al., “The use of oligonucleotides as specifichybridization probes in the Diagnosis of Genetic Disorders,” in HumanGenetic Diseases: A Practical Approach, K. E. Davis Ed., 1986, pp.33-50, IRL: Herndon et al., In Methods in Molecular Biology, White, B.A. Ed., (1993) Vol. 15, pp. 31-39, PCR Protocols: Current Methods andApplications. Humania: Totowa, N.J.).

Generally two short segments of the described sequences can be used inpolymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction can also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from thedescribed nucleic acid fragments, and the sequence of the other primertakes advantage of the presence of the polyadenylic acid tracts to the3′ end of the mRNA precursor encoding microbial genes.

Alternatively, the second primer sequence can be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (e.g., BRL,Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated(Ohara et al., PNAS USA 86:5673, 1989; Loh et al., Science 243:217,1989).

Alternatively, the described acetolactate synthase encoding sequencescan be employed as hybridization reagents for the identification ofhomologs. The basic components of a nucleic acid hybridization testinclude a probe, a sample suspected of containing the gene or genefragment of interest, and a specific hybridization method. Probes aretypically single-stranded nucleic acid sequences that are complementaryto the nucleic acid sequences to be detected. Probes are “hybridizable”to the nucleic acid sequence to be detected. The probe length can varyfrom 5 bases to tens of thousands of bases, and can depend upon thespecific test to be done. Typically a probe length of about 15 bases toabout 30 bases is suitable. However, only part of the probe moleculeneed be complementary to the nucleic acid sequence to be detected. Inaddition, the complementarity between the probe and the target sequenceneed not be perfect. Hybridization does occur between imperfectlycomplementary molecules with the result that a certain fraction of thebases in the hybridized region are not paired with the propercomplementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid can occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness et al., Nucl. Acids Res.19:5143-5151, 1991). Suitable chaotropic agents include, but are notlimited to, guanidinium chloride, guanidinium thiocyanate, sodiumthiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidiumtetrachloroacetate, potassium iodide and cesium trifluoroacetate, amongothers. The chaotropic agent can be present at a final concentration ofabout 3 M. If desired, one can add formamide to the hybridizationmixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives can also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formatssuch as the sandwich assay format. The sandwich assay is particularlyadaptable to hybridization under non-denaturing conditions. A primarycomponent of a sandwich-type assay is a solid support. The solid supporthas adsorbed to it or covalently coupled to it immobilized nucleic acidprobe that is unlabeled and complementary to one portion of thesequence.

Cytosolic expression of acetolactate synthase can be achieved bytransforming with a gene comprising a sequence encoding an acetolactatesynthase protein, with no mitochondrial targeting signal sequence.Methods for gene expression in yeasts are known in the art (see, e.g.,Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecularand Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink(Eds.), Elsevier Academic Press, San Diego, Calif.). Expression of genesin yeast typically requires a promoter, operably linked to a codingregion of interest, and a transcriptional terminator. A number of yeastpromoters can be used in constructing expression cassettes for genesencoding an acetolactate synthase, including, but not limited toconstitutive promoters FBA, GPD1, ADH1, and GPM, and the induciblepromoters GAL1, GAL10, and CUP1. Other yeast promoters include hybridpromoters UAS(PGK1)-FBA1p (SEQ ID NO: 238), UAS(PGK1)-ENO2p (SEQ ID NO:239), UAS(FBA1)-PDClp (SEQ ID NO: 240), UAS(PGK1)-PDClp (SEQ ID NO:241), and UAS(PGK)-OLE1p (SEQ ID NO: 242). Suitable transcriptionalterminators include, but are not limited to FBAt, GPDt, GPMt, ERG10t,GAL1t, CYC1, and ADH1.

Suitable promoters, transcriptional terminators, and coding regions canbe cloned into a yeast 2 micron plasmid and transformed into yeast cells(Ludwig, et al. Gene, 132: 33-40, 1993; US Appl. Pub. No.20080261861A1).

Suitable promoters, transcriptional terminators, and coding regions canbe cloned into E. coli-yeast shuttle vectors, and transformed into yeastcells as described in the Examples. These vectors allow strainpropagation in both E. coli and yeast strains.

Typically the vector contains a selectable marker and sequences allowingautonomous replication or chromosomal integration in the desired host.Typically used plasmids in yeast are shuttle vectors pRS423, pRS424,pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.),which contain an E. coli replication origin (e.g., pMB1), a yeast 2μorigin of replication, and a marker for nutritional selection. Theselection markers for these four vectors are His3 (vector pRS423), Trp1(vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426).Construction of expression vectors with a chimeric gene encoding apolypeptide can be performed by either standard molecular cloningtechniques in E. coli or by the gap repair recombination method in yeast.

The gap repair cloning approach takes advantage of the highly efficienthomologous recombination in yeast. Typically, a yeast vector DNA isdigested (e.g., in its multiple cloning site) to create a “gap” in itssequence. A number of insert DNAs of interest are generated that containa ≧21 by sequence at both the 5′ and the 3′ ends that sequentiallyoverlap with each other, and with the 5′ and 3′ terminus of the vectorDNA. For example, to construct a yeast expression vector for “Gene X,” ayeast promoter and a yeast terminator are selected for the expressioncassette. The promoter and terminator are amplified from the yeastgenomic DNA, and Gene X is either PCR amplified from its source organismor obtained from a cloning vector comprising Gene X sequence. There isat least a 21 by overlapping sequence between the 5′ end of thelinearized vector and the promoter sequence, between the promoter andGene X, between Gene X and the terminator sequence, and between theterminator and the 3′ end of the linearized vector. The “gapped” vectorand the insert DNAs are then co-transformed into a yeast strain andplated on the medium containing the appropriate compound mixtures thatallow complementation of the nutritional selection markers on theplasmids. The presence of correct insert combinations can be confirmedby PCR mapping using plasmid DNA prepared from the selected cells. Theplasmid DNA isolated from yeast (usually low in concentration) can thenbe transformed into an E. coli strain, e.g., TOP10, followed by minipreps and restriction mapping to further verify the plasmid construct.Finally the construct can be verified by sequence analysis.

Like the gap repair technique, integration into the yeast genome alsotakes advantage of the homologous recombination system in yeast.Typically, a cassette containing a coding region plus control elements(promoter and terminator) and auxotrophic marker is PCR-amplified with ahigh-fidelity DNA polymerase using primers that hybridize to thecassette and contain 40-70 base pairs of sequence homology to theregions 5′ and 3′ of the genomic area where insertion is desired. ThePCR product is then transformed into yeast and plated on mediumcontaining the appropriate compound mixtures that allow selection forthe integrated auxotrophic marker. For example, to integrate “Gene X”into chromosomal location “Y”, the promoter-coding region X-terminatorconstruct is PCR amplified from a plasmid DNA construct and joined to anautotrophic marker (such as URA3) by either SOE (splicing by overlapextension) PCR or by common restriction digests and cloning. The fullcassette, containing the promoter-coding region X-terminator-URA3region, is PCR amplified with primer sequences that contain 40-70 by ofhomology to the regions 5′ and 3′ of location “Y” on the yeastchromosome. The PCR product is transformed into yeast and selected ongrowth media lacking uracil. Transformants can be verified either bycolony PCR or by direct sequencing of chromosomal DNA. Alternatively, anintegration vector can be constructed and propagated in E. coli.Elements necessary for chromosomal integration (at least onehost-specific targeting sequence and a yeast selectable marker) can beadded to any suitable E. coli cloning vector. After preparing the vectorfrom the E. coli host, it can be linearized by restriction digestionwithin the host-specific targeting sequence and transformed into yeast.Homologous recombination between the linearized vector and the nativetargeting sequence will result in integration of the entire vector(Rothstein, R., Methods in Enzymology, Vol 194, pp. 281-301).Transformants are obtained by selection for the auxotrophic marker andconfirmed by PCR method or direct sequencing.

In embodiments, the present invention is directed to a method ofproducing a recombinant host cell, comprising transforming a host cellwith (i) at least one polynucleotide encoding a polypeptide thatcatalyzes a substrate to product conversion of a pyruvate-utilizingbiosynthetic pathway; and (ii) a polynucleotide encoding a peptide thatcatalyzes the conversion of pyruvate to acetolactate; wherein thepolynucleotide of (ii) is integrated into the chromosome.

Biosynthetic Pathways

Suitable biosynthetic pathways for production of butanol are known inthe art, and certain suitable pathways are described herein. In someembodiments, the butanol, including isobutanol biosynthetic pathwaycomprises at least one gene that is heterologous to the host cell. Insome embodiments, the butanol biosynthetic pathway comprises more thanone gene that is heterologous to the host cell. In some embodiments, thebutanol biosynthetic pathway comprises heterologous genes encodingpolypeptides corresponding to every step of a biosynthetic pathway. Asused herein heterologous refers to both native and non-native genes thathave been modified for the purposes herein.

Products of pyruvate-utilizing biosynthetic pathway can beadvantageously produced in a host cell of the invention. A list of suchproducts includes, but is not limited to, 2,3-butanediol, 2-butanone,2-butanol, and isobutanol. In embodiments, the pyruvate-utilizingbiosynthetic pathway comprises one or more polynucleotides encoding apolypeptide that catalyzes a substrate to product conversion of thepathway. In embodiments, the one or more polynucleotides are integratedinto a chromosome of the host cell.

In some embodiments, the invention relates to a recombinant host cellcomprising a first heterologous polynucleotide encoding a firstpolypeptide which catalyzes the conversion of a step of apyruvate-utilizing biosynthetic pathway; a second heterologouspolynucleotide encoding a second polypeptide which catalyzes theconversion of a step of a pyruvate-utilizing biosynthetic pathway; and athird heterologous polynucleotide encoding a third polypeptide whichcatalyzes the conversion of a step of a pyruvate-utilizing biosyntheticpathway; wherein the first and second heterologous polynucleotides areintegrated into the chromosome of the host cell; wherein the thirdheterologous polynucleotide is not integrated into the chromosome of thehost cell; and wherein the first, second, and third polypeptidescatalyze different steps of the pyruvate-utilizing biosynthetic pathway.

In some embodiments, the invention relates to a recombinant host cellcomprising (a) a first heterologous polynucleotide encoding a firstpolypeptide which catalyzes a substrate to product conversion ofpyruvate to acetolactate; (b) a second heterologous polynucleotideencoding a second polypeptide which catalyzes the substrate to productconversion of α-ketoisovalerate to isobutyraldehyde; and (c) a thirdheterologous polynucleotide encoding a third polypeptide which catalyzesthe conversion of a step of a isobutanol biosynthetic pathway that isnot the conversion of (a) or (b); wherein the first and secondheterologous polynucleotides are integrated into the chromosome; whereinthe third heterologous polynucleotide is not integrated into thechromosome; and wherein the host cell produces isobutanol.

In some embodiments, the invention relates to a recombinant host cellcomprising (a) a first heterologous polynucleotide encoding a firstpolypeptide which catalyzes a substrate to product conversion ofα-ketoisovalerate to isobutyraldehyde; and (b) a second heterologouspolynucleotide encoding a second polypeptide which catalyzes theconversion of a step of a isobutanol biosynthetic pathway that is notthe conversion of (a); wherein the first heterologous polynucleotide isintegrated into the chromosome; wherein the second heterologouspolynucleotide is not integrated into the chromosome; and wherein thehost cell produces isobutanol.

Biosynthetic pathways starting with a step of converting pyruvate toacetolactate for synthesis of isobutanol are disclosed in U.S. Appl.Pub. No. 20070092957, which is herein incorporated by reference. Asdescribed in U.S. U.S. Appl. Pub. No. 20070092957, steps in an exampleisobutanol biosynthetic pathway using acetolactate include conversionof:

-   -   acetolactate to 2,3-dihydroxyisovalerate (FIG. 2 pathway step b)        as catalyzed for example by acetohydroxy acid isomeroreductase;    -   2,3-dihydroxyisovalerate to α-ketoisovalerate (FIG. 2 pathway        step c) as catalyzed for example by acetohydroxy acid        dehydratase;    -   α-ketoisovalerate to isobutyraldehyde (FIG. 2 pathway step d) as        catalyzed for example by branched-chain α-keto acid        decarboxylase; and    -   isobutyraldehyde to isobutanol (FIG. 2 pathway step e) as        catalyzed for example by branched-chain alcohol dehydrogenase.

Genes and polypeptides that can be used for substrate to productconversions described herein as well as methods of identifying suchgenes and polypeptides, are described herein and/or in the art, forexample, for isobutanol, in the Examples and in U.S. Pat. No. 7,851,188.Ketol-acid reductoisomerase enzymes are described in U.S. Patent Appl.Pub. Nos. 20080261230 A1, 20090163376 A1, 20100197519 A1, and PCT Appl.Pub. No. WO/2011/041415. Examples of KARIs disclosed therein are thosefrom Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAO1, aswell as Pseudomonas fluorescens PF5 mutants. KARIs include Anaerostipescaccae KARI variants “K9G9” and “K9D3” (amino acid sequences SEQ ID NOs:225 and 224, respectively). US Appl. Pub. No. 20100081154 A1, and U.S.Pat. No. 7,851,188 describe dihydroxyacid dehydratases (DHADs),including a DHAD from Streptococcus mutans. Suitable polypeptides tocatalyze the conversion of α-ketoisovalerate to isobutyraldehyde includethose from Listeria grayi, Lactococcus lactis, and Macrococcuscaseolyticus having SEQ ID NOs: 247, 48, and 248, respectively. U.S.Patent Appl. Publ. No. 20090269823 A1 describes SadB, an alcoholdehydrogenase (ADH) from Achromobacter xylosoxidans. Alcoholdehydrogenases also include horse liver ADH and Beijerinkia indica ADH(protein SEQ ID NO: 237). Each of the above-referenced applications andpatents is herein incorporated by reference.

Also described in U.S. Appl. Pub. No. 20070092957 is the construction ofchimeric genes and genetic engineering of yeast, exemplified bySaccharomyces cerevisiae, for isobutanol production using the disclosedbiosynthetic pathways.

In some embodiments, the isobutanol biosynthetic pathway comprises atleast one gene, at least two genes, at least three genes, or at leastfour genes that is/are heterologous to the yeast cell. In someembodiments, the recombinant host cell comprises a heterologous gene foreach substrate to product conversion of an isobutanol biosyntheticpathway. In embodiments, the polypeptide catalyzing the substrate toproduct conversions of acetolactate to 2,3-dihydroxyisovalerate and/orthe polypeptide catalyzing the substrate to product conversion ofisobutyraldehyde to isobutanol are capable of utilizing NADH as acofactor.

Biosynthetic pathways starting with a step of converting pyruvate toacetolactate for synthesis of 2-butanone and 2-butanol are disclosed inU.S. Appl. Pub. Nos. 20070259410 and 20070292927, which are hereinincorporated by reference. A diagram of the disclosed 2-butanone and2-butanol biosynthetic pathways is provided in FIG. 3. 2-Butanone is theproduct made when the last depicted step of converting 2-butanone to2-butanol is omitted. Production of 2-butanone or 2-butanol in a straindisclosed herein benefits from increased availability of acetolactate.As described in U.S. Appl. Pub. No. 20070292927, steps in an examplebiosynthetic pathway using acetolactate include conversion of:

-   -   acetolactate to acetoin (FIG. 3 step b) as catalyzed for example        by acetolactate decarboxylase;    -   acetoin to 2,3-butanediol (FIG. 3 step i) as catalyzed for        example by butanediol dehydrogenase;    -   2,3-butanediol to 2-butanone (FIG. 3 step j) as catalyzed for        example by diol dehydratase or glycerol dehydratase; and    -   2-butanone to 2-butanol (FIG. 3 step f) as catalyzed for example        by butanol dehydrogenase.

Genes that can be used for expression of these enzymes are described inU.S. Appl. Pub. No. 20070292927. The use in this pathway in yeast of thebutanediol dehydratase from Roseburia inulinivorans, RdhtA, (protein SEQID NO:32, coding region SEQ ID NO:31) is disclosed in U.S. Appl. Pub.No. 20090155870. This enzyme is used in conjunction with the butanedioldehydratase reactivase from Roseburia inulinivorans, RdhtB, (protein SEQID NO:34, coding region SEQ ID NO:33).

As described in U.S. Appl. Pub. No. 20070292927, steps in an examplebiosynthetic pathway using acetolactate include conversion of:

-   -   alpha-acetolactate to acetoin (FIG. 3 step b) as catalyzed for        example by acetolactate decarboxylase;    -   acetoin to 3-amino-2-butanol (FIG. 3 step c) as catalyzed for        example by acetoin aminase;    -   3-amino-2-butanol to 3-amino-2-butanol phosphate (FIG. 3 step d)        as catalyzed for example by aminobutanol kinase;    -   3-amino-2-butanol phosphate to 2-butanone (FIG. 3 step e) as        catalyzed for example by aminobutanol phosphate phosphor-lyase;        and    -   2-butanone to 2-butanol (FIG. 3 step f) as catalyzed for example        by butanol dehydrogenase.

2-Butanone is the product made when the last depicted step of converting2-butanone to 2-butanol is omitted. Production of 2-butanone or2-butanol in a strain disclosed herein benefits from increasedavailability of acetolactate.

Useful for the last step of converting 2-butanone to 2-butanol is a newbutanol dehydrogenase isolated from an environmental isolate of abacterium identified as Achromobacter xylosoxidans that is disclosed inU.S. Pub. Appl. No. 20090269823 (DNA: SEQ ID NO:35, protein SEQ IDNO:36).

Also described in U.S. Pub. Appl. No. 20090155870 is the construction ofchimeric genes and genetic engineering of yeast for 2-butanol productionusing the U.S. Appl. Pub. No. 20070292927 disclosed biosyntheticpathway. 2,3-butanediol is an intermediate in this 2-butanol pathway andthe steps in its synthesis are also described above.

In embodiments of the invention, the pyruvate-utilizing biosyntheticpathway forms a product that includes 2,3-butanediol, isobutanol,2-butanol or 2-butanone. In embodiments, the pyruvate-utilizingbiosynthetic pathway is a butanol biosynthetic pathway. In embodiments,the butanol biosynthetic pathway is a 2-butanol biosynthetic pathway oran isobutanol biosynthetic pathway. In embodiments, the host cellcomprises at least one polynucleotide encoding a polypeptide thatcatalyzes a substrate to product conversion of (i) pyruvate toacetolactate; (ii) acetolactate to 2,3-dihydroxyisovalerate; (iii)2,3-dihydroxyisovalerate to α-ketoisovalerate; (iv) α-ketoisovalerate toisobutyraldehyde; or (v) isobutyraldehyde to isobutanol. In embodiments,the host cell comprises at least one polynucleotide encoding apolypeptide that catalyzes a substrate to product conversion of (i)pyruvate to acetolactate; (ii) acetolactate to 2,3-dihydroxyisovalerate;(iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate; (iv)α-ketoisovalerate to isobutyryl-CoA; (v) isobutyryl-CoA toisobutyraldehyde; or (vi) isobutyraldehyde to isobutanol. In otherembodiments, the host cell comprises at least one polynucleotideencoding a polypeptide that catalyzes a substrate to product conversionof (i) pyruvate to acetolactate; (ii) acetolactate to2,3-dihydroxyisovalerate; (iii) 2,3-dihydroxyisovalerate toα-ketoisovalerate; (iv) α-ketoisovalerate to valine; (v) valine toisobutylamine; (vi) isobutylamine to isobutyraldehyde; or (vii)isobutyraldehyde to isobutanol.

In embodiments, the host cell comprises at least one polynucleotideencoding a polypeptide that catalyzes a substrate to product conversionof (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii)acetoin to 2,3-butanediol; or (iv) 2,3-butanediol to 2-butanone. Inembodiments, the host cell comprises at least one polynucleotideencoding a polypeptide that catalyzes a substrate to product conversionof (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii)acetoin to 2,3-butanediol; (iv) 2,3-butanediol to 2-butanone; or (v)2-butanone to 2-butanol.

In embodiments, the recombinant host cell comprises (a) a heterologouspolynucleotide encoding a polypeptide which catalyzes the substrate toproduct conversion of pyruvate to acetolactate, wherein thepolynucleotide is integrated into the chromosome; (b) a heterologouspolynucleotide encoding a polypeptide which catalyzes the substrate toproduct conversion of acetolactate to 2,3-dihydroxyisovalerate; (c) aheterologous polynucleotide encoding a polypeptide which catalyzes thesubstrate to product conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate; and (d) a heterologous polynucleotide encoding apolypeptide which catalyzes the substrate to product conversion ofα-ketoisovalerate to isobutyraldehyde, wherein the host cell issubstantially free of pyruvate decarboxylase activity; and wherein thehost cell produces isobutanol.

In embodiments, the polypeptide which catalyzes the conversion ofacetolactate to 2,3-dihydroxyisovalerate corresponds to the EnzymeCommission Number EC 1.1.1.86. In embodiments, the polypeptide whichcatalyzes the conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate corresponds to the Enzyme Commission Number EC4.2.1.9. In embodiments, the polypeptide which catalyzes the conversionof α-ketoisovalerate to isobutyraldehyde corresponds to the EnzymeCommission Number EC 4.1.1.72 or 4.1.1.1. In other embodiments, thepolypeptide which catalyzes the conversion of isobutyraldehyde toisobutanol corresponds to the Enzyme Commission Number EC 1.1.1.265,1.1.1.1 or 1.1.1.2.

In other embodiments of the invention, one or more of thepolynucleotides encoding a polypeptide which catalyzes the conversion ofany of the biosynthetic pathway steps described herein are on a plasmid.In embodiments, one or more polynucleotides encoding a polypeptide whichcatalyzes the conversion of any of the biosynthetic pathway stepsdescribed herein are integrated into the chromosome at the PDC1-TRX1intergenic region.

In other embodiments, the host cells of the invention can have reducedor substantially eliminated expression of a polypeptide which catalyzesthe conversion of glycerol-3-phosphate into dihydroxyacetone phosphate.In embodiments, the polypeptide which catalyzes the conversion ofglycerol-3-phosphate into dihydroxyacetone phosphate isglycerol-3-phosphate dehydrogenase (GPD). In embodiments, the host cellcomprises a deletion, mutation, and/or substitution in an endogenouspolynucleotide encoding a polypeptide which catalyzes the conversion ofglycerol-3-phosphate into dihydroxyacetone phosphate. In embodiments,the polypeptide which catalyzes the conversion of glycerol-3-phosphateinto dihydroxyacetone phosphate corresponds to Enzyme Commission Number1.1.1.8. In embodiments, the polynucleotide encoding a polypeptide whichcatalyzes the conversion of glycerol-3-phosphate into dihydroxyacetonephosphate is GPD1 or GPD2. In embodiments, the polynucleotide encoding apolypeptide which catalyzes the conversion of glycerol-3-phosphate intodihydroxyacetone phosphate comprises a GPD sequence of Table 2. Suchmodifications and others to host cells are described in US ApplicationPublication No. 20090305363.

In other embodiments, the host cells of the invention can have reducedor substantially eliminated expression of an iron regulatory protein. Inembodiments, the host cells of the invention can have reduced orsubstantially eliminated expression of a polypeptide affectingiron-sulfur (Fe—S) cluster biosynthesis. In embodiments, recombinanthost cells further comprise (a) at least one heterologous polynucleotideencoding a polypeptide having dihydroxy-acid dehydratase activity; and(b)(i) at least one deletion, mutation, and/or substitution in anendogenous gene encoding a polypeptide affecting Fe—S clusterbiosynthesis; and/or (ii) at least one heterologous polynucleotideencoding a polypeptide affecting Fe—S cluster biosynthesis. Inembodiments, the polypeptide affecting Fe—S cluster biosynthesis isencoded by AFT1 (nucleic acid SEQ ID NO: 227, amino acid SEQ ID NO:228), AFT2 (SEQ ID NOs: 229 and 230), FRA2 (SEQ ID NOs: 231 and 232),GRX3(SEQ ID NOs: 233 and 234), or CCC1 (SEQ ID NOs: 235 and 236). Inembodiments, the polypeptide affecting Fe—S cluster biosynthesis isconstitutive mutant AFT1 L99A, AFT1 L102A, AFT1 C291F, or AFT1 C293F. Inembodiments, the polypeptide affecting Fe—S cluster biosynthesis isselected from AFT1, AFT2, PSE1, FRA2, GRX3, MSN5, or combinationsthereof. In embodiments, the host cell comprises a deletion, mutation,and/or substitution in an endogenous polynucleotide encoding an ironregulatory protein. In embodiments, the host cell comprises a deletion,mutation, and/or substitution in an endogenous polynucleotide encoding apolypeptide which affects Fe—S cluster biosynthesis. In embodiments, thepolynucleotide encoding a polypeptide which affects Fe—S clusterbiosynthesis comprises a sequence as disclosed in WIPO Appl. Pub. No.WO/2011/103300.

It will be appreciated that host cells comprising a butanol biosyntheticpathway such as an isobutanol biosynthetic pathway as provided hereinmay further comprise one or more additional modifications. U.S. Appl.Pub. No. 20090305363 (incorporated by reference) discloses increasedconversion of pyruvate to acetolactate by engineering yeast forexpression of a cytosol-localized acetolactate synthase and substantialelimination of pyruvate decarboxylase activity. Modifications to reduceglycerol-3-phosphate dehydrogenase activity and/or disruption in atleast one gene encoding a polypeptide having pyruvate decarboxylaseactivity or a disruption in at least one gene encoding a regulatoryelement controlling pyruvate decarboxylase gene expression as describedin U.S. Patent Appl. Pub. No. 20090305363 (incorporated herein byreference), modifications to a host cell that provide for increasedcarbon flux through an Entner-Doudoroff Pathway or reducing equivalentsbalance as described in U.S. Patent Appl. Pub. No. 20100120105(incorporated herein by reference). Other modifications include at leastone deletion, mutation, and/or substitution in an endogenouspolynucleotide encoding a polypeptide having acetolactate reductaseactivity. In embodiments, the polypeptide having acetolactate reductaseactivity is YMR226C (SEQ ID NO: 226) of Saccharomyces cerevisae or ahomolog thereof. Additional modifications include a deletion, mutation,and/or substitution in an endogenous polynucleotide encoding apolypeptide having aldehyde dehydrogenase and/or aldehyde oxidaseactivity. In embodiments, the polypeptide having aldehyde dehydrogenaseactivity is ALD6 (SEQ ID NO: 223) from Saccharomyces cerevisiae or ahomolog thereof. A genetic modification which has the effect of reducingglucose repression wherein the yeast production host cell is pdc—isdescribed in U.S. Appl. Publication No. 20110124060, incorporated hereinby reference. Additionally, host cells may comprise heterologouspolynucleotides encoding a polypeptide with phosphoketolase activityand/or a heterologous polynucleotide encoding a polypeptide withphosphotransacetylase activity as described in U.S. application Ser. No.13/161,168, filed on Jun. 15, 2011, incorporated herein by reference.Reduced pyruvate decarboxylase activity

Endogenous pyruvate decarboxylase activity in microbial cells convertspyruvate to acetaldehyde, which is then converted to ethanol or toacetyl-CoA via acetate (see FIG. 1). Microbial cells can have one ormore genes encoding pyruvate decarboxylase. For example, in yeast thereis one gene encoding pyruvate decarboxylase in Kluyveromyces lactis,while there are three isozymes of pyruvate decarboxylase encoded by thePDCJ, PDC5, and PDC6 genes in Saccharomyces cerevisiae, as well as apyruvate decarboxylase regulatory gene PDC2. Expression of pyruvatedecarboxylase from PDC6 is minimal. In embodiments of the invention,host cells can have pyruvate decarboxylase activity that is reduced bydisrupting at least one gene encoding a pyruvate decarboxylase, or agene regulating pyruvate decarboxylase gene expression. For example, inS. cerevisiae the PDC1 and PDC5 genes, or all three genes, aredisrupted. In addition, pyruvate decarboxylase activity can be reducedby disrupting the PDC2 regulatory gene in S. cerevisiae. In otheryeasts, genes encoding pyruvate decarboxylase proteins such as thosehaving at least about 80-85%, 85%-90%, 90%-95%, or at least about 98%sequence identity to PDC1 or PDC5 can be disrupted.

Examples of yeast strains with reduced pyruvate decarboxylase activitydue to disruption of pyruvate decarboxylase encoding genes have beenreported such as for Saccharomyces in Flikweert et al. (Yeast,12:247-257, 1996), for Kluyveromyces in Bianchi et al. (Mol. Microbiol.,19(1):27-36, 1996), and disruption of the regulatory gene in Hohmann(Mol Gen Genet., 241:657-666, 1993). Saccharomyces strains having nopyruvate decarboxylase activity are available from the ATCC withAccession #200027 and #200028.

Expression of pyruvate decarboxylase genes can be reduced in any hostcell that is also engineered with acetolactate synthase expression andother biosynthetic pathway enzyme encoding genes for production of acompound derived from acetolactate. Examples of yeast pyruvatedecarboxylase genes that may be targeted for disruption are listed inTable 2 (SEQ ID NOs:50, 52, 54, 56, 58, 60, 62, 64 and 66). Other targetgenes, such as those encoding pyruvate decarboxylase proteins having atleast about 80-85%, 85%-90%, 90%-95%, or at least about 98% or 99%sequence identity to the pyruvate decarboxylases listed in Table 2 (SEQID NOs: 51, 53, 55, 57, 59, 61, 63, 65 and 67) can be identified in theliterature and in bioinformatics databases well known to the skilledperson. Additionally, the sequences described herein or those recited inthe art can be used to identify homologs in other yeast strains, asdescribed above for identification of acetolactate synthase encodinggenes.

Alternatively, because pyruvate decarboxylase encoding sequences arewell known, and because sequencing of the genomes of yeasts isprevalent, suitable pyruvate decarboxylase gene targets can beidentified on the basis of sequence similarity using bioinformaticsapproaches. Genomes have been completely sequenced and annotated and arepublicly available for the following yeast strains: Ashbya gossypii ATCC10895, Candida glabrata CBS 138, Kluyveromyces lactis NRRL Y-1140,Pichia stipitis CBS 6054, Saccharomyces cerevisiae S288c,Schizosaccharomyces pombe 972h-, and Yarrowia hpolytica CLIB122.Typically BLAST (described above) searching of publicly availabledatabases with known pyruvate decarboxylase encoding sequences orpyruvate decarboxylase amino acid sequences, such as those providedherein, is used to identify pyruvate decarboxylase encoding sequences ofother yeasts.

Accordingly it is within the scope of the invention to provide pyruvatedecarboxylase proteins having at least about 70-75%, 75%-80%, 80-85%,85%-90%, 90%-95% or at least about 98% or 99% sequence identity to anyof the pyruvate decarboxylase proteins disclosed herein (SEQ ID NOs:51,53, 55, 57, 59, 61, 63, 65 and 67). Identities are based on the ClustalW method of alignment using the default parameters of GAP PENALTY=10,GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

In embodiments, the host cell of the invention can have expression ofpyruvate decarboxylase, glycerol-3-phosphate dehydrogenase, an ironregulatory protein, and/or a polypeptide affecting iron-sulfur (Fe—S)cluster biosynthesis that is decreased or substantially eliminated. Inother embodiments, the host cell comprises a deletion, mutation, and/orsubstitution in an endogenous polynucleotide encoding a polypeptidehaving the activity of pyruvate decarboxylase, glycerol-3-phosphatedehydrogenase, an iron regulatory protein, or a polypeptide affectingFe—S cluster biosynthesis.

Genes encoding pyruvate decarboxylase, glycerol-3-phosphatedehydrogenase, an iron regulatory protein, or a polypeptide affectingFe—S cluster biosynthesis can be disrupted in any host cell usinggenetic modification. Many methods for genetic modification of targetgenes are known to one skilled in the art and can be used to create thepresent yeast strains. Modifications that can be used to reduce oreliminate expression of a target protein are disruptions that include,but are not limited to, deletion of the entire gene or a portion of thegene, inserting a DNA fragment into the gene (in either the promoter orcoding region) so that the protein is not expressed or expressed atlower levels, introducing a mutation into the coding region which adds astop codon or frame shift such that a functional protein is notexpressed, and introducing one or more mutations into the coding regionto alter amino acids so that a non-functional or a less enzymaticallyactive protein is expressed. In addition, expression of a gene can beblocked by expression of an antisense RNA or an interfering RNA, andconstructs can be introduced that result in cosuppression. Moreover, agene can be synthesized whose expression is low because rare codons aresubstituted for plentiful ones, and this gene substituted for theendogenous gene. Such a gene will produce the same polypeptide but at alower rate. In addition, the synthesis or stability of the transcriptmay be lessened by mutation. Similarly the efficiency by which a proteinis translated from mRNA may be modulated by mutation. All of thesemethods can be readily practiced by one skilled in the art making use ofthe known or identified gene sequences.

DNA sequences surrounding a coding sequence are also useful in somemodification procedures and are available for yeasts such as forSaccharomyces cerevisiae in the complete genome sequence coordinated byGenome Project ID9518 of Genome Projects coordinated by NCBI (NationalCenter for Biotechnology Information) with identifying GOPID #13838.Additional examples of yeast genomic sequences include that of Yarrowialipolytica, GOPIC #13837, and of Candida albicans, which is included inGPID #10771, #10701 and #16373. Other yeast genomic sequences can bereadily found by one of skill in the art in publicly availabledatabases.

In particular, DNA sequences surrounding a gene coding sequence areuseful for modification methods using homologous recombination. Forexample, in this method gene flanking sequences are placed bounding aselectable marker gene to mediate homologous recombination whereby themarker gene replaces the target gene. Also partial gene sequences andgene flanking sequences bounding a selectable marker gene may be used tomediate homologous recombination whereby the marker gene replaces aportion of the target gene. In addition, the selectable marker may bebounded by site-specific recombination sites, so that followingexpression of the corresponding site-specific recombinase, theresistance gene is excised from the target gene locus withoutreactivating the latter. The site-specific recombination leaves behind arecombination site which disrupts expression of the protein. Ahomologous recombination vector can be constructed to also leave adeletion in the target gene following excision of the selectable marker,as is well known to one skilled in the art.

Deletions can be made using mitotic recombination as described in Wachet al. (Yeast, 10:1793-1808, 1994). This method involves preparing a DNAfragment that contains a selectable marker between genomic regions thatcan be as short as 20 bp, and which bound a target DNA sequence. ThisDNA fragment can be prepared by PCR amplification of the selectablemarker gene using as primers oligonucleotides that hybridize to the endsof the marker gene and that include the genomic regions that canrecombine with the yeast genome. The linear DNA fragment can beefficiently transformed into yeast and recombined into the genomeresulting in gene replacement including with deletion of the target DNAsequence (as described in “Methods in Enzymology,” v194, pp 281-301,1991).

In addition, the activity of pyruvate decarboxylase,glycerol-3-phosphate dehydrogenase, an iron regulatory protein, or apolypeptide affecting Fe—S cluster biosynthesis in any host cell of theinvention can be disrupted using random mutagenesis, which is followedby screening to identify strains with reduced pyruvate decarboxylaseactivity. Using this type of method, the DNA sequence of the pyruvatedecarboxylase encoding region, or any other region of the genomeaffecting expression of these activities, need not be known.

Methods for creating genetic mutations are common and well known in theart and may be applied to the exercise of creating mutants. Commonlyused random genetic modification methods (reviewed in Methods in YeastGenetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.) include spontaneous mutagenesis, mutagenesis caused by mutatorgenes, chemical mutagenesis, irradiation with UV or X-rays, ortransposon mutagenesis.

Chemical mutagenesis of yeast commonly involves treatment of cells withone of the following DNA mutagens: ethyl methanesulfonate (EMS), nitrousacid, diethyl sulfate, or N-methyl-N′-nitro-N-nitroso-guanidine (MNNG).These methods of mutagenesis have been reviewed in Spencer et al(Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell andMolecular Biology. Humana Press, Totowa, N.J.). Chemical mutagenesiswith EMS may be performed as described in Methods in Yeast Genetics,2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Irradiation with ultraviolet (UV) light or X-rays can also be used toproduce random mutagenesis in yeast cells. The primary effect ofmutagenesis by UV irradiation is the formation of pyrimidine dimerswhich disrupt the fidelity of DNA replication. Protocols forUV-mutagenesis of yeast can be found in Spencer et al. (Mutagenesis inYeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology.Humana Press, Totowa, N.J.). Introduction of a mutator phenotype canalso be used to generate random chromosomal mutations in yeast. Commonmutator phenotypes can be obtained through disruption of one or more ofthe following genes: PMS1, MAG1, RAD18 or RAD51. Restoration of thenon-mutator phenotype can be easily obtained by insertion of thewildtype allele. Collections of modified cells produced from any ofthese or other known random mutagenesis processes may be screened forreduced activity of pyruvate decarboxylase, glycerol-3-phosphatedecarboxylase, an iron regulatory protein or a polypeptide affectingFe—S cluster biosynthesis.

Host Cells

The host cells of the invention can be any cell amenable to geneticmanipulation.

In embodiments, the host cell can be a bacterium, a cyanobacterium, afilamentous fungus, or a yeast. In embodiments, the host cell is amember of the genus Clostridium, Zymomonas, Escherichia, Salmonella,Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus,Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula, Kluyveromyces, orSaccharomyces. In other embodiments, the host cell is Escherichia coli,Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans,Rhodococcus erythropolis, Pseudomonas putida, Bacillus subtilis,Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium,or Enterococcus faecalis.

Examples of a yeast include, but are not limited to, Saccharomyces,Schizosaccharomyces, Hansenula, Issatchenkia, Candida, Kluyveromyces,Yarrowia and Pichia. Examples of yeast strains include, but are notlimited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces thermotolerans, Candida glabrata,Candida albicans, Pichia stipitis and Yarrowia lipolytica. In someembodiments, the host cell is Saccharomyces cerevisiae. S. cerevisiaeyeast are known in the art and are available from a variety of sources,including, but not limited to, American Type Culture Collection(Rockville, Md.), Centraalbureau voor Schimmelcultures (CBS) FungalBiodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, NorthAmerican Bioproducts, Martrex, and Lallemand. S. cerevisiae include, butare not limited to, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm Pro™yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcoholyeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turboyeast, FerMax™ Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1,PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

In embodiments, the host cell of the invention is a facultativeanaerobe. In embodiments, a cell used as a production host preferablyhas enhanced tolerance to the produced chemical, and/or can have a highrate of carbohydrate utilization. These characteristics can be conferredby mutagenesis and selection, genetic engineering, or can be natural.

Fermentation Media

A host cell of the invention can be grown in fermentation media that cancontain suitable carbon substrates. Suitable substrates can include, butare not limited to, monosaccharides such as glucose and fructose,oligosaccharides such as lactose or sucrose, polysaccharides such asstarch or cellulose or mixtures thereof and unpurified mixtures fromrenewable feedstocks such as cheese whey permeate, cornsteep liquor,sugar beet molasses, and barley malt. Additionally, the carbon substratecan also be one-carbon substrates such as carbon dioxide, or methanolfor which metabolic conversion into key biochemical intermediates hasbeen demonstrated. In addition to one and two carbon substrates,methylotrophic organisms can utilize a number of other carbon containingcompounds such as methylamine, glucosamine and a variety of amino acidsfor metabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993),415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher:Intercept, Andover, UK). Similarly, various species of Candida canmetabolize alanine or oleic acid (Sulter et al., Arch. Microbiol.153:485-489 (1990)). Hence, it is contemplated that the source of carbonutilized in the present invention can encompass a wide variety of carboncontaining substrates.

In addition to a carbon source, fermentation media can contain suitableminerals, salts, cofactors, buffers and other components, known to thoseskilled in the art, suitable for the growth of the cultures andpromotion of the enzymatic pathway necessary for production of thedesired product.

Culture Conditions

Typically host cells of the invention are grown at a temperature in therange of about 20° C. to about 37° C. in an appropriate medium. Suitablegrowth media in the present invention are common commercially preparedmedia such as broth that includes yeast nitrogen base, ammonium sulfate,and dextrose as the carbon/energy source) or YPD Medium, a blend ofpeptone, yeast extract, and dextrose in optimal proportions for growingmost Saccharomyces cerevisiae strains. Other defined or synthetic growthmedia can also be used and the appropriate medium for growth of theparticular microorganism will be known by one skilled in the art ofmicrobiology or fermentation science.

Suitable pH ranges for the fermentation can be between pH 3.0 to pH 7.5.A pH range of pH 4.5.0 to pH 6.5 can be used in an initial condition.

Fermentations can be performed under aerobic or anaerobic conditions,where anaerobic or microaerobic conditions are preferred.

The amount of butanol produced in the fermentation medium can bedetermined using a number of methods known in the art, for example, highperformance liquid chromatography (HPLC) or gas chromatography (GC).

Industrial Batch and Continuous Fermentations

Methods of the present invention can employ a batch method offermentation. A classical batch fermentation is a closed system wherethe composition of the medium is set at the beginning of thefermentation and not subject to artificial alterations during thefermentation. Thus, at the beginning of the fermentation the medium isinoculated with the desired organism or organisms, and fermentation ispermitted to occur without adding anything to the system. Typically,however, a “batch” fermentation is batch with respect to the addition ofcarbon source and attempts are often made at controlling factors such aspH and oxygen concentration. In batch systems, the metabolite andbiomass compositions of the system change constantly up to the time thefermentation is stopped. Within batch cultures cells moderate through astatic lag phase to a high growth log phase and finally to a stationaryphase where growth rate is diminished or halted. If untreated, cells inthe stationary phase will eventually die. Cells in log phase generallyare responsible for the bulk of production of end product orintermediate.

A variation on the standard batch system is the Fed-Batch system.Fed-Batch fermentation processes are also suitable in the presentinvention and comprise a typical batch system with the exception thatthe substrate is added in increments as the fermentation progresses.Fed-Batch systems are useful when catabolite repression is apt toinhibit the metabolism of the cells and where it is desirable to havelimited amounts of substrate in the media. Measurement of the actualsubstrate concentration in Fed-Batch systems is difficult and istherefore estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and Fed-Batch fermentations are common and well knownin the art and examples may be found in Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, Second Edition(1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, MukundV., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated byreference.

Additionally, the methods of the present invention can be adaptable tocontinuous fermentation methods. Continuous fermentation is an opensystem where a defined fermentation medium is added continuously to abioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous fermentation generallymaintains the cultures at a constant high density where cells areprimarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or end product concentration.For example, one method will maintain a limiting nutrient such as thecarbon source or nitrogen level at a fixed rate and allow all otherparameters to moderate. In other systems a number of factors affectinggrowth can be altered continuously while the cell concentration,measured by media turbidity, is kept constant. Continuous systems striveto maintain steady state growth conditions and thus the cell loss due tothe medium being drawn off must be balanced against the cell growth ratein the fermentation. Methods of modulating nutrients and growth factorsfor continuous fermentation processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

It is contemplated that the present invention can be practiced usingeither batch, fed-batch or continuous processes and that any known modeof fermentation would be suitable. Additionally, it is contemplated thatcells may be immobilized on a substrate as whole cell catalysts andsubjected to fermentation conditions for butanol production.

Methods for Product Isolation from the Fermentation Medium

Products of the biosynthetic pathways of the invention (e.g., butanol)can be isolated from the fermentation medium using methods known in theart. For example, solids can be removed from the fermentation medium byextraction, centrifugation, filtration, decantation, or the like. Then,the product (e.g., butanol) can be isolated from the fermentationmedium, which has been treated to remove solids as described above,using methods such as distillation, liquid-liquid extraction, ormembrane-based separation. Because butanol forms a low boiling point,azeotropic mixture with water, distillation can only be used to separatethe mixture up to its azeotropic composition. Distillation can be usedin combination with another separation method to obtain separationaround the azeotrope. Methods that can be used in combination withdistillation to isolate and purify butanol include, but are not limitedto, decantation, liquid-liquid extraction, adsorption, andmembrane-based techniques. Additionally, butanol can be isolated usingazeotropic distillation using an entrainer (see for example Doherty andMalone, Conceptual Design of Distillation Systems, McGraw Hill, NewYork, 2001).

The butanol-water mixture forms a heterogeneous azeotrope so thatdistillation can be used in combination with decantation to isolate andpurify the butanol. In this method, the butanol containing fermentationbroth is distilled to near the azeotropic composition. Then, theazeotropic mixture is condensed, and the butanol is separated from thefermentation medium by decantation. The decanted aqueous phase can bereturned to the first distillation column as reflux. The butanol-richdecanted organic phase can be further purified by distillation in asecond distillation column.

The products such as butanol can also be isolated from the fermentationmedium using liquid-liquid extraction in combination with distillation.In this method, the butanol is extracted from the fermentation brothusing liquid-liquid extraction with a suitable solvent. Thebutanol-containing organic phase is then distilled to separate thebutanol from the solvent.

Distillation in combination with adsorption can also be used to isolatebutanol from a fermentation medium. In this method, the fermentationbroth containing the butanol is distilled to near the azeotropiccomposition and then the remaining water is removed by use of anadsorbent, such as molecular sieves (Aden et al., LignocellulosicBiomass to Ethanol Process Design and Economics Utilizing Co-CurrentDilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover,Report NREL/TP-510-32438, National Renewable Energy Laboratory, June2002).

Additionally, distillation in combination with pervaporation can be usedto isolate and purify a product such as butanol from the fermentationmedium. In this method, the fermentation broth containing the butanol isdistilled to near the azeotropic composition, and then the remainingwater is removed by pervaporation through a hydrophilic membrane (Guo etal., J. Membr. Sci. 245:199-210, 2004).

Methods for producing and recovering a product such as butanol from afermentation broth using extractive fermentation are described in detailin U.S. patent application Ser. No. 12/478,389 filed on Jun. 4, 2009 andcorresponding published U.S. Appn. Publ. No. 20090305370, U.S.Provisional Appl. No. 61/231,699 filed on Aug. 6, 2009, U.S. ProvisionalAppl. No. 61/368,429 filed on Jul. 28, 2010, and U.S. Appn. Publ. Nos.20100221802 and 20110097773. Such methods include those which comprisethe step of contacting the fermentation broth with a water immiscibleorganic extractant selected from the group consisting of C₁₂ to C₂₂fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fattyacids, C₁₂ to C₂₂ fatty amides, C₁₂ to C₂₂ fatty aldehydes, and mixturesthereof, to form a two-phase mixture comprising an aqueous phase and abutanol-containing organic phase. “Contacting” means the fermentationmedium and the organic extractant are brought into physical contact atany time during the fermentation process.

Examples of suitable extractants include, but are not limited to, anextractant comprising at least one solvent selected from the groupconsisting of oleyl alcohol, behenyl alcohol, cetyl alcohol, laurylalcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid,myristic acid, stearic acid, methyl myristate, methyl oleate, lauricaldehyde, 1-nonanol, 1-decanol, 1-undecanol, 2-undecanol, 1-nonanal, andmixtures thereof. In one embodiment, the extractant comprises oleylalcohol. These organic extractants are available commercially fromvarious sources, such as Sigma-Aldrich (St. Louis, Mo.), in variousgrades, many of which are suitable for use in extractive fermentation toproduce or recover butanol. Technical grades contain a mixture ofcompounds, including the desired component and higher and lower fattycomponents. For example, one commercially available technical gradeoleyl alcohol contains about 65% oleyl alcohol and a mixture of higherand lower fatty alcohols.

In embodiments, the present invention is directed to a method ofproducing butanol, comprising (a) providing a recombinant host cell ofthe invention; and (b) contacting the host cell with a fermentablecarbon substrate to form a fermentation broth under conditions wherebybutanol is produced. In other embodiments, the method further comprisescontacting the fermentation broth with an extractant to produce atwo-phase fermentation mixture. In other embodiments, the extractantcomprises fatty acids. In other embodiments, the fatty acids are derivedfrom corn oil or soybean oil. In other embodiments, the extractantcomprises a water immiscible organic extractant selected from the groupconsisting of: C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, estersof C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, C₁₂ to C₂₂ fattyamides. In other embodiments, the method further comprises contactingthe fermentation broth with an organic acid and an enzyme capable ofesterifying the butanol with the organic acid. In embodiments, themethod further comprises vaporizing at least a portion of thefermentation broth to form a vapor stream comprising water and butanol.

Methods for measuring butanol titer and production are known. Forexample, butanol titer and production can be measured using gaschromatography (GC) or high performance liquid chromatography (HPLC) asdescribed in the examples. In embodiments, the amount of butanolproduced by a host cell of the invention is increased by at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 2-fold, atleast about 3-fold, or at least about 4-fold greater as compared to theamount of butanol produced by a host cell that does not comprise apolynucleotide encoding a polypeptide that catalyzes the conversion ofpyruvate to acetolactate integrated into the chromosome. In embodiments,the titer of butanol produced by a host cell of the invention isincreased by at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 2-fold, at least about 3-fold, or at least about 4-foldgreater as compared to a recombinant host cell wherein thepolynucleotide encoding a polypeptide that catalyzes the conversion ofpyruvate to acetolactate is not integrated into the chromosome.

In embodiments, the present invention is directed to a method forincreasing the copy number or expression of a non-integrated recombinantpolynucleotide encoding a polypeptide that catalyzes a step of abiosynthetic pathway described herein, comprising contacting a host cellof the invention with a fermentable carbon substrate to form afermentation broth under conditions whereby the product of thebiosynthetic pathway is produced, such as the fermentation conditionsdescribed herein. In other embodiments, the present invention isdirected to a method for increasing the flux in a pyruvate-utilizingbiosynthetic pathway, comprising contacting a host cell of the inventionwith a fermentable carbon substrate to form a fermentation broth underconditions whereby the flux in the pyruvate-utilizing biosyntheticpathway in the host cell is increased, such as the fermentationconditions described herein.

In other embodiments, the invention is directed to a method ofincreasing the formation of a product of a pyruvate-utilizingbiosynthetic pathway comprising (i) providing a recombinant host cell ofthe invention; and (ii) growing the host cell under conditions whereinthe product of the pyruvate-utilizing pathway is formed, wherein theamount of product formed by the recombinant host cell is greater thanthe amount of product formed by a host cell that does not comprise apolynucleotide encoding a polypeptide which catalyzes the conversion ofpyruvate to acetolactate integrated into the chromosome. In otherembodiments, the pyruvate-utilizing biosynthetic pathway forms2,3-butanediol, isobutanol, 2-butanol or 2-butanone. In otherembodiments, the pyruvate-utilizing biosynthetic pathway is a butanolbiosynthetic pathway. In other embodiments, the butanol biosyntheticpathway is (a) a 2-butanol biosynthetic pathway; or (b) an isobutanolbiosynthetic pathway.

In other embodiments, the invention is directed to a compositioncomprising (i) a host cell of the invention; (ii) butanol; and (iii) anextractant. In other embodiments, the invention is directed to acomposition comprising (i) a host cell of the invention; (ii) butanol;(iii) an extractant; and (iv) an esterification enzyme. Anesterification enzyme is one that catalyzes the reaction between andacid and an alcohol to generate an ester. In the broadest senseesterfication enzymes are hydrolases that act on an ester linkage andoften referred to as esterases. As used herein lipases, are a subclassof esterases shown to be effective in forming esters between the fattyacids and isobutanol present in the broth. Such lipases may include oneor more esterase enzymes, for example, hydrolase enzymes such as lipaseenzymes. Lipase enzymes used may be derived from any source, including,for example, Absidia, Achromobacter, Aeromonas, Alcaligenes, Alternaria,Aspergillus, Achromobacter, Aureobasidium, Bacillus, Beauveria,Brochothrix, Candida, Chromobacter, Coprinus, Fusarium, Geotricum,Hansenula, Humicola, Hyphozyma, Lactobacillus, Metarhizium, Mucor,Nectria, Neurospora, Paecilomyces, Penicillium, Pseudomonas,Rhizoctonia, Rhizomucor, Rhizopus, Rhodosporidium, Rhodotorula,Saccharomyces, Sus, Sporobolomyces, Thermomyces, Thiarosporella,Trichoderma, Verticillium, and/or a strain of Yarrowia. In a preferredaspect, the source of the lipase is selected from the group consistingof Absidia blakesleena, Absidia corymbifera, Achromobacter iophagus,Alcaligenes sp., Alternaria brassiciola, Aspergillus flavus, Aspergillusniger, Aureobasidium pullulans, Bacillus pumilus, Bacillusstrearothermophilus, Bacillus subtilis, Brochothrix thermosohata,Candida cylindracea (Candida rugosa), Candida paralipolytica, CandidaAntarctica lipase A, Candida antartica lipase B, Candida ernobii,Candida deformans, Chromobacter viscosum, Coprinus cinerius, Fusariumoxysporum, Fusarium solani, Fusarium solani pisi, Fusarium roseumculmorum, Geotricum penicillatum, Hansenula anomala, Humicolabrevispora, Humicola brevis var. thermoidea, Humicola insolens,Lactobacillus curvatus, Rhizopus oryzae, Penicillium cyclopium,Penicillium crustosum, Penicillium expansum, Penicillium sp. I,Penicillium sp. II, Pseudomonas aeruginosa, Pseudomonas alcaligenes,Pseudomonas cepacia (syn. Burkholderia cepacia), Pseudomonasfluorescens, Pseudomonas fragi, Pseudomonas maltophilia, Pseudomonasmendocina, Pseudomonas mephitica lipolytica, Pseudomonas alcaligenes,Pseudomonas plantari, Pseudomonas pseudoalcaligenes, Pseudomonas putida,Pseudomonas stutzeri, and Pseudomonas wisconsinensis, Rhizoctoniasolani, Rhizomucor miehei, Rhizopus japonicus, Rhizopus microsporus,Rhizopus nodosus, Rhodosporidium toruloides, Rhodotorula glutinis,Saccharomyces cerevisiae, Sporobolomyces shibatanus, Sus scrofa,Thermomyces lanuginosus (formerly Humicola lanuginose), Thiarosporellaphaseolina, Trichoderma harzianum, Trichoderma reesei, and Yarrowialipolytica. In a further preferred aspect, the lipase is selected fromthe group consisting of Thermomcyces lanuginosus, Aspergillus sp.lipase, Aspergillus niger lipase, Candida antartica lipase B,Pseudomonas sp. lipase, Penicillium roqueforti lipase, Penicilliumcamembertii lipase, Mucor javanicus lipase, Burkholderia cepacia lipase,Alcaligenes sp. lipase, Candida rugosa lipase, Candida parapsilosislipase, Candida deformans lipase, lipases A and B from Geotrichumcandidum, Neurospora crassa lipase, Nectria haematococca lipase,Fusarium heterosporum lipase Rhizopus delemar lipase, Rhizomucor mieheilipase, Rhizopus arrhizus lipase, and Rhizopus oryzae lipase. Suitablecommercial lipase preparations suitable as enzyme catalyst 42 include,but are not limited to Lipolase® 100 L, Lipex® 100 L, Lipoclean® 2000T,Lipozyme® CALB L, Novozym® CALA L, and Palatase 20000 L, available fromNovozymes, or from Pseudomonas fluorescens, Pseudomonas cepacia, Mucormiehei, hog pancreas, Candida cylindracea, Rhizopus niveus, Candidaantarctica, Rhizopus arrhizus or Aspergillus available fromSigmaAldrich.

In embodiments, the extractant comprises fatty acids. In embodiments,the fatty acids are derived from corn oil or soybean oil. In otherembodiments, the extractant is a water immiscible organic extractant. Inother embodiments, the extractant is C₁₂ to C₂₂ fatty alcohols, C₁₂ toC₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, or C₁₂ to C₂₂ fattyaldehydes.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook et al.,Molecular Cloning: A Laboratory Manual; Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989) (Maniatis), Silhavy et al.,Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1984), Ausubel et al., Current Protocols inMolecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987), and by Methods in Yeast Genetics, 2005, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt et al., eds,American Society for Microbiology, Washington, D C., 1994) or by ThomasD. Brock in Biotechnology: A Textbook of Industrial Microbiology (SecondEdition, Sinauer Associates, Inc., Sunderland, Mass., 1989). Allreagents, restriction enzymes and materials used for the growth andmaintenance of microbial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), LifeTechnologies (Rockville, Md.), New England Biolabs (Ipswich, Mass.) orSigma Chemical Company (St. Louis, Mo.) unless otherwise specified.Microbial strains were obtained from The American Type CultureCollection (ATCC), Manassas, Va., unless otherwise noted. All theoligonucleotide primers were synthesized by Sigma-Genosys (Woodlands,Tex.) or Integrated DNA Technologies (Coralsville, Iowa). Syntheticcomplete medium is described in Amberg, Burke and Strathern, 2005,Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

GC

The GC method utilized an HP-InnoWax column (30 m×0.32 mm ID, 0.25 μmfilm) from Agilent Technologies (Santa Clara, Calif.). The carrier gaswas helium at a flow rate of 1 ml/min measured at 150° C. with constanthead pressure; injector split was 1:10 at 200° C.; oven temperature was45° C. for 1 min, 45° C. to 230° C. at 10° C./min, and 230° C. for 30sec. FID detection was used at 260° C. with 40 ml/min helium makeup gas.Culture broth samples were filtered through 0.2 μM spin filters beforeinjection. Depending on analytical sensitivity desired, either 0.1 μm or0.5 μm injection volumes were used. Calibrated standard curves weregenerated for the following compounds: ethanol, isobutanol, acetoin,meso-2,3-butanediol, and (2S,3S)-2,3-butanediol. Analytical standardswere also utilized to identify retention times for isobutryaldehyde,isobutyric acid, and isoamyl alcohol.

HPLC

Analysis for fermentation by-product composition is well known to thoseskilled in the art. For example, one high performance liquidchromatography (HPLC) method utilizes a Shodex SH-1011 column with aShodex SH-G guard column (both available from Waters Corporation,Milford, Mass.), with refractive index (RI) detection. Chromatographicseparation is achieved using 0.01 M H₂SO₄ as the mobile phase with aflow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanolretention time is 47.6 minutes.

Methods for Determining Isobutanol Concentration in Culture Media

The concentration of isobutanol in the culture media can be determinedby a number of methods known in the art. For example, a specific highperformance liquid chromatography (HPLC) method utilized a ShodexSH-1011 column with a Shodex SH-G guard column, both purchased fromWaters Corporation (Milford, Mass.), with refractive index (RI)detection. Chromatographic separation was achieved using 0.01 M H₂SO₄ asthe mobile phase with a flow rate of 0.5 mL/min and a column temperatureof 50° C. Isobutanol had a retention time of 46.6 min under theconditions used. Alternatively, gas chromatography (GC) methods areavailable. For example, a specific GC method utilized an HP-INNOWaxcolumn (30 m×0.53 mm id, 1 μm film thickness, Agilent Technologies,Wilmington, Del.), with a flame ionization detector (FID). The carriergas was helium at a flow rate of 4.5 mL/min, measured at 150° C. withconstant head pressure; injector split was 1:25 at 200° C.; oventemperature was 45° C. for 1 min, 45 to 220° C. at 10° C./min, and 220°C. for 5 min; and FID detection was employed at 240° C. with 26 mL/minhelium makeup gas. The retention time of isobutanol was 4.5 min.

The meaning of abbreviations is as follows: “s” means second(s), “min”means minute(s), “h” means hour(s), “psi” means pounds per square inch,“nm” means nanometers, “d” means day(s), “μL” means microliter(s), “mL”means milliliter(s), “L” means liter(s), “mm” means millimeter(s), “nm”means nanometers, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmol” means micromole(s), “g” means gram(s), “μg” meansmicrogram(s) and “ng” means nanogram(s), “PCR” means polymerase chainreaction, “OD” means optical density, “OD₆₀₀” means the optical densitymeasured at a wavelength of 600 nm, “kDa” means kilodaltons, “g” meansthe gravitation constant, “bp” means base pair(s), “kbp” means kilobasepair(s), “% w/v” means weight/volume percent, “% v/v” meansvolume/volume percent, “wt %” means percent by weight, “HPLC” means highperformance liquid chromatography, and “GC” means gas chromatography.The term “molar selectivity” is the number of moles of product producedper mole of sugar substrate consumed and is reported as a percent.

Example 1

Construction of Saccharomyces cerevisiae strain BP1083 (“NGCI-070”;PNY1504)

The strain BP1064 was derived from CEN.PK 113-7D (CBS 8340;Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,Netherlands) and contains deletions of the following genes: URA3, HIS3,PDC1, PDC5, PDC6, and GPD2. BP1064 was transformed with plasmids pYZ090(SEQ ID NO:134) and pLH468 (SEQ ID NO:135) to create strain NGCI-070(BP1083, PNY1504).

Deletions, which completely removed the entire coding sequence, werecreated by homologous recombination with PCR fragments containingregions of homology upstream and downstream of the target gene andeither a G418 resistance marker or URA3 gene for selection oftransformants. The G418 resistance marker, flanked by loxP sites, wasremoved using Cre recombinase. The URA3 gene was removed by homologousrecombination to create a scarless deletion, or if flanked by loxP siteswas removed using Cre recombinase.

The scarless deletion procedure was adapted from Akada et al., Yeast,23:399, 2006. In general, the PCR cassette for each scarless deletionwas made by combining four fragments, A-B-U-C, by overlapping PCR. ThePCR cassette contained a selectable/counter-selectable marker, URA3(Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, alongwith the promoter (250 by upstream of the URA3 gene) and terminator (150by downstream of the URA3 gene). Fragments A and C, each 500 by long,corresponded to the 500 by immediately upstream of the target gene(Fragment A) and the 3′ 500 by of the target gene (Fragment C).Fragments A and C were used for integration of the cassette into thechromosome by homologous recombination. Fragment B (500 by long)corresponded to the 500 by immediately downstream of the target gene andwas used for excision of the URA3 marker and Fragment C from thechromosome by homologous recombination, as a direct repeat of thesequence corresponding to Fragment B was created upon integration of thecassette into the chromosome. Using the PCR product ABUC cassette, theURA3 marker was first integrated into and then excised from thechromosome by homologous recombination. The initial integration deletedthe gene, excluding the 3′ 500 bp. Upon excision, the 3′ 500 by regionof the gene was also deleted. For integration of genes using thismethod, the gene to be integrated was included in the PCR cassettebetween fragments A and B.

URA3 Deletion

To delete the endogenous URA3 coding region, a ura3::loxP-kanMX-loxPcassette was PCR-amplified from pLA54 template DNA (SEQ ID NO:136).pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and isflanked by loxP sites to allow recombination with Cre recombinase andremoval of the marker. PCR was done using Phusion DNA polymerase andprimers BK505 and BK506 (SEQ ID NOs:137 and 138). The URA3 portion ofeach primer was derived from the 5′ region upstream of the URA3 promoterand 3′ region downstream of the coding region such that integration ofthe loxP-kanMX-loxP marker resulted in replacement of the URA3 codingregion. The PCR product was transformed into CEN.PK 113-7D usingstandard genetic techniques (Methods in Yeast Genetics, 2005, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202)and transformants were selected on YPD containing G418 (100 μg/ml) at30° C. Transformants were screened to verify correct integration by PCRusing primers LA468 and LA492 (SEQ ID NOs:139 and 140) and designatedCEN.PK 113-7D Δura3::kanMX.

HIS3 Deletion

The four fragments for the PCR cassette for the scarless HIS3 deletionwere amplified using Phusion High Fidelity PCR Master Mix (New EnglandBioLabs; Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template,prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia,Calif.). HIS3 Fragment A was amplified with primer oBP452 (SEQ IDNO:141) and primer oBP453 (SEQ ID NO:142), containing a 5′ tail withhomology to the 5′ end of HIS3 Fragment B. HIS3 Fragment B was amplifiedwith primer oBP454 (SEQ ID NO:143), containing a 5′ tail with homologyto the 3′ end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO:144),containing a 5′ tail with homology to the 5′ end of HIS3 Fragment U.HIS3 Fragment U was amplified with primer oBP456 (SEQ ID NO:145),containing a 5′ tail with homology to the 3′ end of HIS3 Fragment B, andprimer oBP457 (SEQ ID NO:146), containing a 5′ tail with homology to the5′ end of HIS3 Fragment C. HIS3 Fragment C was amplified with primeroBP458 (SEQ ID NO:147), containing a 5′ tail with homology to the 3′ endof HIS3 Fragment U, and primer oBP459 (SEQ ID NO:148). PCR products werepurified with a PCR Purification kit (Qiagen). HIS3 Fragment AB wascreated by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment Band amplifying with primers oBP452 (SEQ ID NO:141) and oBP455 (SEQ IDNO:144). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQID NO:145) and oBP459 (SEQ ID NO:148). The resulting PCR products werepurified on an agarose gel followed by a Gel Extraction kit (Qiagen).The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQID NO:141) and oBP459 (SEQ ID NO:148). The PCR product was purified witha PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::kanMX were made and transformedwith the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast TransformationII kit (Zymo Research; Orange, Calif.). Transformation mixtures wereplated on synthetic complete media lacking uracil supplemented with 2%glucose at 30° C. Transformants with a his3 knockout were screened forby PCR with primers oBP460 (SEQ ID NO:149) and oBP461 (SEQ ID NO:150)using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit(Qiagen). A correct transformant was selected as strain CEN.PK 113-7DΔura3::kanMX Δhis3::URA3.

KanMX Marker Removal from the ΔUra3 Site and URA3 Marker Removal fromthe Δhis3 Site

The KanMX marker was removed by transforming CEN.PK 113-7D Δura3::kanMXΔhis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 66, described in U.S.Provisional Appl. No. 61/290,639) using a Frozen-EZ Yeast TransformationII kit (Zymo Research) and plating on synthetic complete medium lackinghistidine and uracil supplemented with 2% glucose at 30° C.Transformants were grown in YP supplemented with 1% galactose at 30° C.for ˜6 hours to induce the Cre recombinase and KanMX marker excision andplated onto YPD (2% glucose) plates at 30° C. for recovery. An isolatewas grown overnight in YPD and plated on synthetic complete mediumcontaining 5-fluoro-orotic acid (0.1%) at 30° C. to select for isolatesthat lost the URA3 marker. 5-FOA resistant isolates were grown in andplated on YPD for removal of the pRS423::PGAL1-cre plasmid. Isolateswere checked for loss of the KanMX marker, URA3 marker, andpRS423::PGAL1-cre plasmid by assaying growth on YPD+G418 plates,synthetic complete medium lacking uracil plates, and synthetic completemedium lacking histidine plates. A correct isolate that was sensitive toG418 and auxotrophic for uracil and histidine was selected as strainCEN.PK 113-7D Δura3::loxP Δhis3 and designated as BP857. The deletionsand marker removal were confirmed by PCR and sequencing with primersoBP450 (SEQ ID NO:151) and oBP451 (SEQ ID NO:152) for Δura3 and primersoBP460 (SEQ ID NO:149) and oBP461 (SEQ ID NO:150) for Δhis3 usinggenomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen).

PDC6 Deletion

The four fragments for the PCR cassette for the scarless PDC6 deletionwere amplified using Phusion High Fidelity PCR Master Mix (New EnglandBioLabs) and CEN.PK 113-7D genomic DNA as template, prepared with aGentra Puregene Yeast/Bact kit (Qiagen). PDC6 Fragment A was amplifiedwith primer oBP440 (SEQ ID NO:153) and primer oBP441 (SEQ ID NO:154),containing a 5′ tail with homology to the 5′ end of PDC6 Fragment B.PDC6 Fragment B was amplified with primer oBP442 (SEQ ID NO:155),containing a 5′ tail with homology to the 3″ end of PDC6 Fragment A, andprimer oBP443 (SEQ ID NO:156), containing a 5′ tail with homology to the5′ end of PDC6 Fragment U. PDC6 Fragment U was amplified with primeroBP444 (SEQ ID NO:157), containing a 5′ tail with homology to the 3′ endof PDC6 Fragment B, and primer oBP445 (SEQ ID NO:158), containing a 5′tail with homology to the 5′ end of PDC6 Fragment C. PDC6 Fragment C wasamplified with primer oBP446 (SEQ ID NO:159), containing a 5′ tail withhomology to the 3′ end of PDC6 Fragment U, and primer oBP447 (SEQ IDNO:160). PCR products were purified with a PCR Purification kit(Qiagen). PDC6 Fragment AB was created by overlapping PCR by mixing PDC6Fragment A and PDC6 Fragment B and amplifying with primers oBP440 (SEQID NO:153) and oBP443 (SEQ ID NO:156). PDC6 Fragment UC was created byoverlapping PCR by mixing PDC6 Fragment U and PDC6 Fragment C andamplifying with primers oBP444 (SEQ ID NO:157) and oBP447 (SEQ IDNO:160). The resulting PCR products were purified on an agarose gelfollowed by a Gel Extraction kit (Qiagen). The PDC6 ABUC cassette wascreated by overlapping PCR by mixing PDC6 Fragment AB and PDC6 FragmentUC and amplifying with primers oBP440 (SEQ ID NO:153) and oBP447 (SEQ IDNO:160). The PCR product was purified with a PCR Purification kit(Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 were made andtransformed with the PDC6 ABUC PCR cassette using a Frozen-EZ YeastTransformation II kit (Zymo Research). Transformation mixtures wereplated on synthetic complete media lacking uracil supplemented with 2%glucose at 30° C. Transformants with a pdc6 knockout were screened forby PCR with primers oBP448 (SEQ ID NO:161) and oBP449 (SEQ ID NO:162)using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit(Qiagen). A correct transformant was selected as strain CEN.PK 113-7DΔura3::loxP Δhis3 Δpdc6::URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3 was grown overnight in YPDand plated on synthetic complete medium containing 5-fluoro-orotic acid(0.1%) at 30° C. to select for isolates that lost the URA3 marker. Thedeletion and marker removal were confirmed by PCR and sequencing withprimers oBP448 (SEQ ID NO:161) and oBP449 (SEQ ID NO:162) using genomicDNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absenceof the PDC6 gene from the isolate was demonstrated by a negative PCRresult using primers specific for the coding sequence of PDC6, oBP554(SEQ ID NO:163) and oBP555 (SEQ ID NO:164). The correct isolate wasselected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 and designatedas BP891.

PDC1 Deletion ilvDSm Integration

The PDC1 gene was deleted and replaced with the ilvD coding region fromStreptococcus mutans ATCC #700610. The A fragment followed by the ilvDcoding region from Streptococcus mutans for the PCR cassette for thePDC1 deletion-ilvDSm integration was amplified using Phusion HighFidelity PCR Master Mix (New England BioLabs) and NYLA83 (described inU.S. Provisional Appl. No. 61/246,709) genomic DNA as template, preparedwith a Gentra Puregene Yeast/Bact kit (Qiagen). PDC1 Fragment A-ilvDSm(SEQ ID NO:165) was amplified with primer oBP513 (SEQ ID NO:166) andprimer oBP515 (SEQ ID NO:167), containing a 5′ tail with homology to the5′ end of PDC1 Fragment B. The B, U, and C fragments for the PCRcassette for the PDC1 deletion-ilvDSm integration were amplified usingPhusion High Fidelity PCR Master Mix (New England BioLabs) and CEN.PK113-7D genomic DNA as template, prepared with a Gentra PuregeneYeast/Bact kit (Qiagen). PDC1 Fragment B was amplified with primeroBP516 (SEQ ID NO:168) containing a 5′ tail with homology to the 3′ endof PDC1 Fragment A-ilvDSm, and primer oBP517 (SEQ ID NO:169), containinga 5′ tail with homology to the 5′ end of PDC1 Fragment U. PDC1 FragmentU was amplified with primer oBP518 (SEQ ID NO:170), containing a 5′ tailwith homology to the 3′ end of PDC1 Fragment B, and primer oBP519 (SEQID NO:171), containing a 5′ tail with homology to the 5′ end of PDC1Fragment C. PDC1 Fragment C was amplified with primer oBP520 (SEQ IDNO:172), containing a 5′ tail with homology to the 3′ end of PDC1Fragment U, and primer oBP521 (SEQ ID NO:173). PCR products werepurified with a PCR Purification kit (Qiagen). PDC1 Fragment A-ilvDSm-Bwas created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm and PDC1Fragment B and amplifying with primers oBP513 (SEQ ID NO:166) and oBP517(SEQ ID NO:169). PDC1 Fragment UC was created by overlapping PCR bymixing PDC1 Fragment U and PDC1 Fragment C and amplifying with primersoBP518 (SEQ ID NO:170) and oBP521 (SEQ ID NO:173). The resulting PCRproducts were purified on an agarose gel followed by a Gel Extractionkit (Qiagen). The PDC1 A-ilvDSm-BUC cassette (SEQ ID NO:174) was createdby overlapping PCR by mixing PDC1 Fragment A-ilvDSm-B and PDC1 FragmentUC and amplifying with primers oBP513 (SEQ ID NO:166) and oBP521 (SEQ IDNO:173). The PCR product was purified with a PCR Purification kit(Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 were made andtransformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZYeast Transformation II kit (Zymo Research). Transformation mixtureswere plated on synthetic complete media lacking uracil supplemented with2% glucose at 30 C. Transformants with a pdc1 knockout ilvDSmintegration were screened for by PCR with primers oBP511 (SEQ ID NO:175)and oBP512 (SEQ ID NO:176) using genomic DNA prepared with a GentraPuregene Yeast/Bact kit (Qiagen). The absence of the PDC1 gene from theisolate was demonstrated by a negative PCR result using primers specificfor the coding sequence of PDC1, oBP550 (SEQ ID NO:177) and oBP551 (SEQID NO:178). A correct transformant was selected as strain CEN.PK 113-7DΔura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3 was grownovernight in YPD and plated on synthetic complete medium containing5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lostthe URA3 marker. The deletion of PDC1, integration of ilvDSm, and markerremoval were confirmed by PCR and sequencing with primers oBP511 (SEQ IDNO:175) and oBP512 (SEQ ID NO:176) using genomic DNA prepared with aGentra Puregene Yeast/Bact kit (Qiagen). The correct isolate wasselected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmand designated as BP907.

PDC5 Deletion sadB Integration

The PDC5 gene was deleted and replaced with the sadB coding region fromAchromobacter xylosoxidans. A segment of the PCR cassette for the PDC5deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS.

pUC19-URA3MCS is pUC19 based and contains the sequence of the URA3 genefrom Saccharomyces cerevisiae situated within a multiple cloning site(MCS). pUC19 contains the pMB1 replicon and a gene coding forbeta-lactamase for replication and selection in Escherichia coli. Inaddition to the coding sequence for URA3, the sequences from upstreamand downstream of this gene were included for expression of the URA3gene in yeast. The vector can be used for cloning purposes and can beused as a yeast integration vector.

The DNA encompassing the URA3 coding region along with 250 by upstreamand 150 by downstream of the URA3 coding region from Saccharomycescerevisiae CEN.PK 113-7D genomic DNA was amplified with primers oBP438(SEQ ID NO:179), containing BamHI, AscI, PmeI, and FseI restrictionsites, and oBP439 (SEQ ID NO:180), containing XbaI, Pad, and NotIrestriction sites, using Phusion High-Fidelity PCR Master Mix (NewEngland BioLabs). Genomic DNA was prepared using a Gentra PuregeneYeast/Bact kit (Qiagen). The PCR product and pUC19 (SEQ ID NO:181) wereligated with T4 DNA ligase after digestion with BamHI and XbaI to createvector pUC19-URA3MCS. The vector was confirmed by PCR and sequencingwith primers oBP264 (SEQ ID NO:182) and oBP265 (SEQ ID NO:183).

The coding sequence of sadB and PDC5 Fragment B were cloned intopUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCRcassette. The coding sequence of sadB was amplified using pLH468-sadB(SEQ ID NO:184) as template with primer oBP530 (SEQ ID NO:185),containing an AscI restriction site, and primer oBP531 (SEQ ID NO:186),containing a 5′ tail with homology to the 5′ end of PDC5 Fragment B.PDC5 Fragment B was amplified with primer oBP532 (SEQ ID NO:187),containing a 5′ tail with homology to the 3′ end of sadB, and primeroBP533 (SEQ ID NO:188), containing a PmeI restriction site. PCR productswere purified with a PCR Purification kit (Qiagen). sadB-PDC5 Fragment Bwas created by overlapping PCR by mixing the sadB and PDC5 Fragment BPCR products and amplifying with primers oBP530 (SEQ ID NO:185) andoBP533 (SEQ ID NO:188). The resulting PCR product was digested with AscIand PmeI and ligated with T4 DNA ligase into the corresponding sites ofpUC19-URA3MCS after digestion with the appropriate enzymes. Theresulting plasmid was used as a template for amplification ofsadB-Fragment B-Fragment U using primers oBP536 (SEQ ID NO:189) andoBP546 (SEQ ID NO:190), containing a 5′ tail with homology to the 5′ endof PDC5 Fragment C. PDC5 Fragment C was amplified with primer oBP547(SEQ ID NO:191) containing a 5′ tail with homology to the 3′ end of PDC5sadB-Fragment B-Fragment U, and primer oBP539 (SEQ ID NO:192). PCRproducts were purified with a PCR Purification kit (Qiagen). PDC5sadB-Fragment B-Fragment U-Fragment C was created by overlapping PCR bymixing PDC5 sadB-Fragment B-Fragment U and PDC5 Fragment C andamplifying with primers oBP536 (SEQ ID NO:189) and oBP539 (SEQ IDNO:192). The resulting PCR product was purified on an agarose gelfollowed by a Gel Extraction kit (Qiagen). The PDC5 A-sadB-BUC cassette(SEQ ID NO:193) was created by amplifying PDC5 sadB-Fragment B-FragmentU-Fragment C with primers oBP542 (SEQ ID NO:194), containing a 5′ tailwith homology to the 50 nucleotides immediately upstream of the nativePDC5 coding sequence, and oBP539 (SEQ ID NO:192). The PCR product waspurified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmwere made and transformed with the PDC5 A-sadB-BUC PCR cassette using aFrozen-EZ Yeast Transformation II kit (Zymo Research). Transformationmixtures were plated on synthetic complete media lacking uracilsupplemented with 1% ethanol (no glucose) at 30 C. Transformants with apdc5 knockout sadB integration were screened for by PCR with primersoBP540 (SEQ ID NO:195) and oBP541 (SEQ ID NO:196) using genomic DNAprepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence ofthe PDC5 gene from the isolate was demonstrated by a negative PCR resultusing primers specific for the coding sequence of PDC5, oBP552 (SEQ IDNO:197) and oBP553 (SEQ ID NO:198). A correct transformant was selectedas strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmΔpdc5::sadB-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3 wasgrown overnight in YPE (1% ethanol) and plated on synthetic completemedium supplemented with ethanol (no glucose) and containing5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost theURA3 marker. The deletion of PDC5, integration of sadB, and markerremoval were confirmed by PCR with primers oBP540 (SEQ ID NO:195) andoBP541 (SEQ ID NO:196) using genomic DNA prepared with a Gentra PuregeneYeast/Bact kit (Qiagen). The correct isolate was selected as strainCEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB anddesignated as BP913.

GPD2 Deletion

To delete the endogenous GPD2 coding region, a gpd2::loxP-URA3-loxPcassette (SEQ ID NO:131) was PCR-amplified using loxP-URA3-loxP PCR (SEQID NO:200) as template DNA. loxP-URA3-loxP contains the URA3 marker from(ATCC #77107) flanked by loxP recombinase sites. PCR was done usingPhusion DNA polymerase and primers LA512 and LA513 (SEQ ID NOs:201 and202). The GPD2 portion of each primer was derived from the 5′ regionupstream of the GPD2 coding region and 3′ region downstream of thecoding region such that integration of the loxP-URA3-loxP markerresulted in replacement of the GPD2 coding region. The PCR product wastransformed into BP913 and transformants were selected on syntheticcomplete media lacking uracil supplemented with 1% ethanol (no glucose).Transformants were screened to verify correct integration by PCR usingprimers oBP582 and AA270 (SEQ ID NOs:198 and 203).

The URA3 marker was recycled by transformation with pRS423::PGAL1-cre(SEQ ID NO:204) and plating on synthetic complete media lackinghistidine supplemented with 1% ethanol at 30 C. Transformants werestreaked on synthetic complete medium supplemented with 1% ethanol andcontaining 5-fluoro-orotic acid (0.1%) and incubated at 30 C to selectfor isolates that lost the URA3 marker. 5-FOA resistant isolates weregrown in YPE (1% ethanol) for removal of the pRS423::PGAL1-cre plasmid.The deletion and marker removal were confirmed by PCR with primersoBP582 (SEQ ID NO:198) and oBP591 (SEQ ID NO:205). The correct isolatewas selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6Δpdc1::ilvDSm Δpdc5::sadB Δgpd2::loxP and designated as BP1064(PNY1503).

BP1064 was transformed with plasmids pYZ090 (SEQ ID NO:134) and pLH468(SEQ ID NO:135) to create strain NGCI-070 (BP1083; PNY1504).

pYZ090 is based on the pHR81 (ATCC #87541, Manassas, Va.) backbone andwas constructed to contain a chimeric gene having the coding region ofthe alsS gene from Bacillus subtilis (nt position 457-2172) expressedfrom the yeast CUP1 promoter (nt 2-449) and followed by the CYC1terminator (nt 2181-2430) for expression of ALS, and a chimeric genehaving the coding region of the ilvC gene from Lactococcus lactis (nt3634-4656) expressed from the yeast ILV5 promoter (2433-3626) andfollowed by the ILV5 terminator (nt 4682-5304) for expression of KARI.The pLH468 plasmid (SEQ ID NO:2) was constructed for expression of DHAD,KivD and HADH in yeast and is described in U.S. Application PublicationNo. 20090305363, herein incorporated by reference.

Example 2 Construction of Saccharomyces cerevisiae Strains BP1135 andPNY1507 and Isobutanol-Producing Derivatives

The purpose of this Example was to construct Saccharomyces cerevisiaestrains BP1135 and PNY1507. These strains were derived from PNY1503(BP1064). PNY1503 was derived from CEN.PK 113-7D (CBS 8340;Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,Netherlands). The construction of PNY1503 (BP1064) is described above.BP1135 contains an additional deletion of the FRA2 gene. PNY1507 wasderived from BP1135 with additional deletion of the ADH1 gene, withintegration of the kivD gene from Lactococcus lactis, codon optimizedfor expression in Saccharomyces cerevisiae, into the ADH1 locus.

Deletions, which generally removed the entire coding sequence, werecreated by homologous recombination with PCR fragments containingregions of homology upstream and downstream of the target gene and theURA3 gene for selection of transformants. The URA3 gene was removed byhomologous recombination to create a scarless deletion. Geneintegrations were generated in a similar manner.

The scarless deletion procedure was adapted from Akada et al., Yeast,23:399, 2006. In general, the PCR cassette for each scarless deletionwas made by combining four fragments, A-B-U-C, by overlapping PCR. Insome instances, the individual fragments were first cloned into aplasmid prior to the entire cassette being amplified by PCR for thedeletion/integration procedure. The PCR cassette contained aselectable/counter-selectable marker, URA3 (Fragment U), consisting ofthe native CEN.PK 113-7D URA3 gene, along with the promoter (250 byupstream of the URA3 gene) and terminator (150 by downstream of the URA3gene) regions. Fragments A and C, each generally 500 by long,corresponded to the 500 by immediately upstream of the target gene(Fragment A) and the 3′ 500 by of the target gene (Fragment C).Fragments A and C were used for integration of the cassette into thechromosome by homologous recombination. Fragment B (500 by long)corresponded to the 500 by immediately downstream of the target gene andwas used for excision of the URA3 marker and Fragment C from thechromosome by homologous recombination, as a direct repeat of thesequence corresponding to Fragment B was created upon integration of thecassette into the chromosome.

Using the PCR product ABUC cassette, the URA3 marker was firstintegrated into and then excised from the chromosome by homologousrecombination. The initial integration deleted the gene, excluding the3′ 500 bp. Upon excision, the 3′ 500 by region of the gene was alsodeleted. For integration of genes using this method, the gene to beintegrated was included in the PCR cassette between fragments A and B.

FRA2 Deletion

The FRA2 deletion was designed to delete 250 nucleotides from the 3′ endof the coding sequence, leaving the first 113 nucleotides of the FRA2coding sequence intact. An in-frame stop codon was present 7 nucleotidesdownstream of the deletion. The four fragments for the PCR cassette forthe scarless FRA2 deletion were amplified using Phusion High FidelityPCR Master Mix (New England BioLabs; Ipswich, Mass.) and CEN.PK 113-7Dgenomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit(Qiagen; Valencia, Calif.). FRA2 Fragment A was amplified with primeroBP594 (SEQ ID NO:99) and primer oBP595 (SEQ ID NO:100), containing a 5′tail with homology to the 5′ end of FRA2 Fragment B. FRA2 Fragment B wasamplified with primer oBP596 (SEQ ID NO:101), containing a 5′ tail withhomology to the 3′ end of FRA2 Fragment A, and primer oBP597 (SEQ IDNO:102), containing a 5′ tail with homology to the 5′ end of FRA2Fragment U. FRA2 Fragment U was amplified with primer oBP598 (SEQ IDNO:103), containing a 5′ tail with homology to the 3′ end of FRA2Fragment B, and primer oBP599 (SEQ ID NO:104), containing a 5′ tail withhomology to the 5′ end of FRA2 Fragment C. FRA2 Fragment C was amplifiedwith primer oBP600 (SEQ ID NO:105), containing a 5′ tail with homologyto the 3′ end of FRA2 Fragment U, and primer oBP601 (SEQ ID NO:106). PCRproducts were purified with a PCR Purification kit (Qiagen). FRA2Fragment AB was created by overlapping PCR by mixing FRA2 Fragment A andFRA2 Fragment B and amplifying with primers oBP594 (SEQ ID NO:99) andoBP597 (SEQ ID NO:102). FRA2 Fragment UC was created by overlapping PCRby mixing FRA2 Fragment U and FRA2 Fragment C and amplifying withprimers oBP598 (SEQ ID NO:103) and oBP601 (SEQ ID NO:106). The resultingPCR products were purified on an agarose gel followed by a GelExtraction kit (Qiagen). The FRA2 ABUC cassette was created byoverlapping PCR by mixing FRA2 Fragment AB and FRA2 Fragment UC andamplifying with primers oBP594 (SEQ ID NO:99 and oBP601 (SEQ ID NO:106).The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of PNY1503 were made and transformed with the FRA2 ABUCPCR cassette using a Frozen-EZ Yeast Transformation II kit (ZymoResearch; Orange, Calif.). Transformation mixtures were plated onsynthetic complete media lacking uracil supplemented with 1% ethanol at30° C. Transformants with a fra2 knockout were screened for by PCR withprimers oBP602 (SEQ ID NO:107) and oBP603 (SEQ ID NO:108) using genomicDNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). A correcttransformant was grown in YPE (yeast extract, peptone, 1% ethanol) andplated on synthetic complete medium containing 5-fluoro-orotic acid(0.1%) at 30° C. to select for isolates that lost the URA3 marker. Thedeletion and marker removal were confirmed by PCR with primers oBP602(SEQ ID NO:107) and oBP603 (SEQ ID NO:108) using genomic DNA preparedwith a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the FRA2gene from the isolate was demonstrated by a negative PCR result usingprimers specific for the deleted coding sequence of FRA2, oBP605 (SEQ IDNO:109) and oBP606 (SEQ ID NO:110). The correct isolate was selected asstrain CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t pdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5tgpd2Δ::loxP fra2Δ and designated as PNY1505 (BP1135). This strain wastransformed with isobutanol pathway plasmids (pYZ090, SEQ ID NO:134) andpLH468 (U.S. Provisional Appl. No. 61/246,709, filed Sep. 29, 2009), andone clone was designated BP1168 (PNY1506).

ADH1 Deletion and kivD Ll(y) Integration

The ADH1 gene was deleted and replaced with the kivD coding region fromLactococcus lactis codon optimized for expression in Saccharomycescerevisiae. The scarless cassette for the ADH1 deletion-kivD_Ll(y)integration was first cloned into plasmid pUC19-URA3MCS, as described inU.S. Appln. No. 61/356,379, filed Jun. 18, 2010, incorporated herein byreference.

The kivD coding region from Lactococcus lactis codon optimized forexpression in Saccharomyces cerevisiae was amplified using pLH468 (U.S.Provisional Appl. No. 61/246,709, filed Sep. 29, 2009) as template withprimer oBP562 (SEQ ID NO:111), containing a PmeI restriction site, andprimer oBP563 (SEQ ID NO:112), containing a 5′ tail with homology to the5′ end of ADH1 Fragment B. ADH1 Fragment B was amplified from genomicDNA prepared as above with primer oBP564 (SEQ ID NO:113), containing a5′ tail with homology to the 3′ end of kivD_Ll(y), and primer oBP565(SEQ ID NO:114), containing a FseI restriction site. PCR products werepurified with a PCR Purification kit (Qiagen). kivD_Ll(y)-ADH1 FragmentB was created by overlapping PCR by mixing the kivD_Ll(y) and ADH1Fragment B PCR products and amplifying with primers oBP562 (SEQ IDNO:111) and oBP565 (SEQ ID NO:114). The resulting PCR product wasdigested with PmeI and FseI and ligated with T4 DNA ligase into thecorresponding sites of pUC19-URA3MCS after digestion with theappropriate enzymes. ADH1 Fragment A was amplified from genomic DNA withprimer oBP505 (SEQ ID NO:115), containing a SacI restriction site, andprimer oBP506 (SEQ ID NO:116), containing an AscI restriction site. TheADH1 Fragment A PCR product was digested with SacI and AscI and ligatedwith T4 DNA ligase into the corresponding sites of the plasmidcontaining kivD_Ll(y)-ADH1 Fragment B. ADH1 Fragment C was amplifiedfrom genomic DNA with primer oBP507 (SEQ ID NO:117), containing a Padrestriction site, and primer oBP508 (SEQ ID NO:118), containing a SalIrestriction site. The ADH1 Fragment C PCR product was digested with Padand SalI and ligated with T4 DNA ligase into the corresponding sites ofthe plasmid containing ADH1 Fragment A-kivD_Ll(y)-ADH1 Fragment B. Thehybrid promoter UAS(PGK1)-P_(FBA1) was amplified from vectorpRS316-UAS(PGK1)-P_(FBA1)-GUS (described below; SEQ ID NO:206) withprimer oBP674 (SEQ ID NO:119), containing an AscI restriction site, andprimer oBP675 (SEQ ID NO:120), containing a PmeI restriction site. TheUAS(PGK1)-P_(FBA1) PCR product was digested with AscI and PmeI andligated with T4 DNA ligase into the corresponding sites of the plasmidcontaining kivD_Ll(y)-ADH1 Fragments ABC. The entire integrationcassette was amplified from the resulting plasmid with primers oBP505(SEQ ID NO:115) and oBP508 (SEQ ID NO:118) and purified with a PCRPurification kit (Qiagen).

Competent cells of PNY1505 were made and transformed with theADH1-kivD_Ll(y) PCR cassette constructed above using a Frozen-EZ YeastTransformation II kit (Zymo Research). Transformation mixtures wereplated on synthetic complete media lacking uracil supplemented with 1%ethanol at 30° C. Transformants were grown in YPE (1% ethanol) andplated on synthetic complete medium containing 5-fluoro-orotic acid(0.1%) at 30° C. to select for isolates that lost the URA3 marker. Thedeletion of ADH1 and integration of kivD_Ll(y) were confirmed by PCRwith external primers oBP495 (SEQ ID NO:121) and oBP496 (SEQ ID NO:122)and with kivD_Ll(y) specific primer oBP562 (SEQ ID NO:111) and externalprimer oBP496 (SEQ ID NO:122) using genomic DNA prepared with a GentraPuregene Yeast/Bact kit (Qiagen). The correct isolate was selected asstrain CEN.PK 113-7D MATa ura3Δ::loxP his3Δ pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5tgpd2Δ::loxP fra2Δ adh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t anddesignated as PNY1507 (BP1201). PNY1507 was transformed with isobutanolpathway plasmids pYZ090 (SEQ ID NO:134) and pBP915 (described below).Isobutanol production by these derivatives is described below.

Construction of the pRS316-UAS(PGK1)-FBA1p-GUS Vector

To clone a cassette UAS(PGK1)-FBA1p (SEQ ID NO:129), first a 602 bp FBA1promoter (FBA1p) was PCR-amplified from genomic DNA of CEN.PK withprimers T-FBA1(SalI) (SEQ ID NO:123) and B-FBA1(SpeI) (SEQ ID NO:124),and cloned into SalI and SpeI sites on the plasmid pWS358-PGK1p-GUS (SEQID NO:130) after the PGK1p promoter was removed with a SalI/SpeI digestof the plasmid, yielding pWS358-FBA1p-GUS. The pWS358-PGK1p-GUS plasmidwas generated by inserting a PGK1p and beta-glucuronidase gene (GUS) DNAfragments into multiple cloning site of pWS358, which was derived frompRS423 vector (Christianson et al., Gene, 110:119-122, 1992). Secondly,the resulting pWS358-FBA1p-GUS plasmid was digested with SalI and SacI,a DNA fragment containing a FBA1p promoter, GUS gene, and FBAtterminator gel-purified, and cloned into SalI/SacI sites on pRS316 tocreate pRS316-FBA1p-GUS. Thirdly, a 118 bp DNA fragment containing anupstream activation sequence (UAS) located between positions −519 and−402 upstream of the 3-phosphoglycerate kinase (PGK1) open readingframe, namely UAS(PGK1), was PCR-amplified from genomic DNA of CEN.PKwith primers T-U/PGK1(KpnI) (SEQ ID NO:125) and B-U/PGK1(SalI) (SEQ IDNO:126). The PCR product was digested with KpnI and SalI and cloned intoKpnI/SalI sites on pRS316-FBA1p-GUS to createpRS316-UAS(PGK1)-FBA1p-GUS.

Example 3 Construction of PNY2204 and Isobutanol-Producing Derivatives

The purpose of this example is to describe construction of a vector toenable integration of a gene encoding acetolactate synthase into thenaturally occurring intergenic region between the PDC1 and TRX1 codingsequences in Chromosome XII.

Construction of Integration Vector pUC19-kan::pdc1::FBA-alsS::TRX1

The FBA-alsS-CYCt cassette was constructed by moving the 1.7 kbBbvCI/PacI fragment from pRS426::GPD::alsS::CYC (U.S. Appl. Pub. No.20070092957) to pRS426::FBA::ILV5::CYC (U.S. Appl. Pub. No. 20070092957,previously digested with BbvCI/PacI to release the ILV5 gene). Ligationreactions were transformed into E. coli TOP10 cells and transformantswere screened by PCR using primers N98SeqF1 (SEQ ID NO:91) and N99SeqR2(SEQ ID NO:93). The FBA-alsS-CYCt cassette was isolated from the vectorusing BglII and NotI for cloning into pUC19-URA3::ilvD-TRX1 (asdescribed in U.S. Appln. No. 61/356,379, filed Jun. 18, 2010,incorporated herein by reference, clone “B”; herein SEQ ID NO: 243) atthe AflII site (Klenow fragment was used to make ends compatible forligation). Transformants containing the alsS cassette in bothorientations in the vector were obtained and confirmed by PCR usingprimers N98SeqF4 (SEQ ID NO:92) and N1111 (SEQ ID NO:97) forconfiguration “A” and N98SeqF4 (SEQ ID NO:92) and N1110 (SEQ ID NO:96)for configuration “B”. A geneticin selectable version of the “A”configuration vector was then made by removing the URA3 gene (1.2 kbNotI/NaeI fragment) and adding a geneticin cassette (SEQ ID NO: 244herein; previously described in U.S. Appln. No. 61/356,379, filed Jun.18, 2010, incorporated herein by reference) maintained in a pUC19 vector(cloned at the SmaI site). The kan gene was isolated from pUC19 by firstdigesting with KpnI, removal of 3′ overhanging DNA using Klenow Fragment(NEB, Cat. No. M212), digesting with HincII and then gel purifying the1.8 kb gene fragment (Zymoclean™ Gel DNA Recovery Kit, Cat. No. D4001,Zymo Research, Orange, Calif.; SEQ ID NO: 245). Klenow fragment was usedto make all ends compatible for ligation, and transformants werescreened by PCR to select a clone with the geneticin resistance gene inthe same orientation as the previous URA3 marker using primers BK468(SEQ ID NO:90) and N160SeqF5 (SEQ ID NO:94). The resulting clone wascalled pUC19-kan::pdc1::FBA-alsS::TRX1 (clone A)(SEQ ID NO:131).

Construction of alsS Integrant Strains and Isobutanol-ProducingDerivatives

The pUC19-kan::pdc1::FBA-alsS integration vector described above waslinearized with PmeI and transformed into PNY1507 (described above inExample 1). PmeI cuts the vector within the cloned pdc1-TRX1 intergenicregion and thus leads to targeted integration at that location (RodneyRothstein, Methods in Enzymology, 1991, volume 194, pp. 281-301).Transformants were selected on YPE plus 50 μg/ml G418. Patchedtransformants were screened by PCR for the integration event usingprimers N160SeqF5 (SEQ ID NO:94) and oBP512 (SEQ ID NO:98). Twotransformants were tested indirectly for acetolactate synthase functionby evaluating the strains ability to make isobutanol. To do this,additional isobutanol pathway genes were supplied on E. coli-yeastshuttle vectors (pYZ090ΔalsS and pBP915, described below). One clone,strain MATa ura3Δ::loxP his3Δ pdc6Δpdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-pUC19-loxP-kanMX-loxP-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δadh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t was designated as PNY2204. Theplasmid-free parent strain was designated PNY2204. The PNY2204 locus(pdc1Δ::ilvD::pUC19-kan::FBA-alsS::TRX1) is depicted in FIG. 5.

Isobutanol Pathway Plasmids (pYZ090ΔalsS and pBP915)

pYZ090 (SEQ ID NO:134) was digested with SpeI and NotI to remove most ofthe CUP1 promoter and all of the alsS coding sequence and CYCterminator. The vector was then self-ligated after treatment with Klenowfragment and transformed into E. coli Stbl3 cells, selecting forampicillin resistance. Removal of the DNA region was confirmed for twoindependent clones by DNA sequencing across the ligation junction by PCRusing primer N191 (SEQ ID NO:95). The resulting plasmid was namedpYZ090ΔalsS (SEQ ID NO:132).

pBP915 was constructed from pLH468 (SEQ ID NO:124) by deleting the kivDgene and 957 base pairs of the TDH3 promoter upstream of kivD. pLH468was digested with SwaI and the large fragment (12896 bp) was purified onan agarose gel followed by a Gel Extraction kit (Qiagen; Valencia,Calif.). The isolated fragment of DNA was self-ligated with T4 DNAligase and used to transform electrocompetent TOP10 Escherichia coli(Invitrogen; Carlsbad, Calif.). Plasmids from transformants wereisolated and checked for the proper deletion by restriction analysiswith the SwaI restriction enzyme. Isolates were also sequenced acrossthe deletion site with primers oBP556 (SEQ ID NO:127) and oBP561 (SEQ IDNO:128). A clone with the proper deletion was designated pBP915(pLH468ΔkivD)(SEQ ID NO:133).

Example 4 Isobutanol Production in Strains with an Integrated Copy ofthe kivD Gene

The purpose of this example is to show isobutanol production in strainswith an integrated copy of the kivD gene compared to strains withplasmid-borne kivD. Strains without the kivD integration, carryingplasmids pYZ090 and pLH468, were compared to the integration strain,PNY1507, carrying plasmid pYZ090 and pBP915. All media components werefrom Sigma-Aldrich, St. Louis, Mo. Strains were grown in syntheticmedium (Yeast Nitrogen Base Without Amino Acids and Yeast SyntheticDrop-Out Media Supplement without uracil, histidine, tryptophan, andleucine) supplemented with 76 mg/L tryptophan, 380 mg/L leucine, 100 mMMES pH5.5, 20 mg/L nicotinic acid, 20 mg/L thiamine hydrochloride, 0.2%glucose, and 0.2% ethanol. Overnight cultures were grown in 8 ml ofmedium in 125 ml vented Erlenmeyer flasks at 30° C., 250 RPM in a NewBrunswick Scientific 124 shaker. 19 mL of medium in 125 mLtightly-capped Erlenmeyer flasks was inoculated with overnight cultureto an OD600 0.5 and grown for 8 hours at 30° C., 250 RPM in a NewBrunswick Scientific 124 shaker. Glucose was added to 2% (time 0 hours).After 48 hours, culture supernatants (collected using Spin-X centrifugetube filter units, Costar Cat. No. 8169) were analyzed by HPLC permethods described in U.S. Appl. Pub. No. 20070092957. Results are shownin Table 3. The strains with an integrated copy of the kivD gene has asimilar isobutanol titer compared to strains with plasmid-borne kivD.

TABLE 3 Isobutanol titer in strains with an integrated or plasmid-bornekivD gene Strain Isobutanol Titer (g/L) PNY1506 (BP1168) 1.7 +/− 0.3 (n= 2*) PNY1507/pYZ090/pBP915 1.8 +/− 0.1 (n = 2^(#)) *Biologicalreplicates ^(#)Independent transformants

Example 5 Isobutanol Production in Strains with an Integrated Copy ofthe alsS Gene

The purpose of this example is to show increased production ofisobutanol when the acetolactate synthase was removed from a plasmid andintegrated into the yeast genome. Strains without alsS integration(PNY1507 carrying plasmids pYZ090 and pBP915) were compared to theintegration strains (PNY2204 carrying plasmid pYZ090DalsS and pBP915).All strains were grown in synthetic complete medium, minus histidine anduracil containing 0.3% glucose and 0.3% ethanol as carbon sources (10 mLmedium in 125 mL vented Erlenmeyer flasks (VWR Cat. No. 89095-260).After overnight incubation (30° C., 250 rpm in an Innova®40 NewBrunswick Scientific Shaker), cultures were diluted back to 0.2 OD(Eppendorf BioPhotometer measurement) in synthetic complete mediumcontaining 2% glucose and 0.05% ethanol (20 ml medium in 125 mLtightly-capped Erlenmeyer flasks (VWR Cat. No. 89095-260)). After 48hours incubation (30° C., 250 rpm in an Innova®40 New BrunswickScientific Shaker), culture supernatants (collected using Spin-Xcentrifuge tube filter units, Costar Cat. No. 8169) were analyzed byHPLC per methods described in U.S. Appl. Pub. No. 20070092957. Resultsare shown below in Table 4. The isobutanol titer from strains with anintegrated copy of the alsS gene were significantly greater than theisobutanol titer without alsS integration.

TABLE 4 Isobutanol titer in strains with or without alsS geneintegration Strain Isobutanol Titer (g/L) PNY1507/pYZ090/pBP915 1.5 +/−0.2 (n = 3*) PNY2204/pYZ090ΔalsS/pBP915 2.6 +/− 0.1 (n = 3*) (PNY2205)*Biological replicates

Example 6 Isobutanol Production in Strains with an Integrated Copy ofthe alsS Gene

The purpose of this Example is to show increased cell density andproduction of isobutanol when the acetolactate synthase was removed froma plasmid and integrated into the yeast genome. Strains without alsSintegration (PNY1504 as described in U.S. Appln. No. 61/379,546, filedSep. 2, 2010, incorporated herein by reference, and PNY1506) werecompared to the integration strain PNY2205 (PNY2204 transformed withpYZ090ΔalsS and pBP915 plasmids and having alsS integration).

Inoculum and Bioreactor Media

A yeast inoculum media (1 L) was prepared containing 6.7 g of YeastNitrogen Base w/o amino acids (Difco 0919-15-3); 2.8 g Yeast SyntheticDrop-out Medium Supplement Without Histidine, Leucine, Tryptophan andUracil (Sigma Y2001); 20 mL of 1% (w/v) L-Leucine; 4 mL of 1% (w/v)L-Tryptophan; 0.8 mL of Ergosterol & Tween solution; 3 g of ethanol; and3 g of glucose. For 10 mL Ergosterol & Tween solution, 100 mg ofErgosterol was dissolved in 5 mL 100% ethanol and 5 mL Tween 80. Thesolution was heated for 10 min at 70° C.

A 125 mL shake flask was inoculated directly from a frozen vial bypipetting the whole vial culture (approx. 1 ml) into 10 mL of theinoculum medium. The flask was incubated at 260 rpm and 30° C. Thestrain was grown overnight until OD about 1.0. OD at λ=600 nm wasdetermined in a HEXIOS a spectrophotometer (Thermo Electron Corporation,USA). At this point, a 2 L shake flask containing 110 mL of the inoculummedium were inoculated from the overnight culture. The starting OD inthe 2 L flask was 0.1. The flask was incubated at 260 rpm and 30° C.When OD in the shake flask reached about 1.0, 20 mL of 1M MES buffer, 20mL of 10×yeast extract and peptone (YEP), glucose up to finalconcentration of 30 g/L and about 160 mL of oleyl alcohol (90-95%,Cognis, Cincinnati Ohio, USA) were added to the shake flask. 24 hoursafterwards, the oleyl alcohol was removed and bioreactors inoculated.

A 10×YEP solution was prepared by dissolving 100 g of yeast extract and200 of peptone in water to a final volume of 1 L.

A bioreactor medium (1 L) was prepared containing:

(i) salts: ammonium sulfate 5.0 g, potassium phosphate monobasic 2.8 g,magnesium sulfate heptahydrate 1.9 g, zinc sulfate heptahydrate 0.2 g;(ii) vitamins: biotin (D−) 0.40 mg, Ca D(+) panthotenate 8.00 mg,myo-inositol 200.00 mg, pyridoxol hydrochloride 8.00 mg, p-aminobenzoicacid 1.60 mg, riboflavin 1.60 mg, folic acid 0.02 mg, niacin 30.0 mg,and thiamine 30 mg;(iii) amino acids: yeast synthetic drop-out medium supplement withouthistidine, leucine, tryptophan and uracil (Sigma Y2001) 2.8 g, 1% (w/v)L-leucine 20 mL, and 1% (w/v) L-tryptophan 4 mL; and(iv) trace elements: EDTA (Titriplex 1117) 99.38 mg, zinc sulphateheptahydrate 29.81 mg, manganese chloride dehydrate 5.57 mg,cobalt(II)chloride hexahydrate 1.99 mg, copper(II)sulphate pentahydrate1.99 mg, Di-sodium molybdenum dehydrate 2.65 mg, calcium chloridedehydrate 29.81 mg, iron sulphate heptahydrate 19.88 mg, boric acid.

Bioreactor Experimental Design

Experiments were executed in 2 L BIOSTAT B-DCU Tween2 L bioreactors fromSartorius (USA). The fermentors are connected to mass-spec from ThermoElectron Corporation (USA) Directly after inoculation with 80 mL of theinoculum the volume in fermentors was about 800 mL, dissolved oxygentension (DOT) was controlled at 10%, pH was controlled at 5.25, aerationwas controlled at 0.5 L/min, 0.8 L of oleyl alcohol was added. Oleylalcohol was used in order to extract isobutanol from culture broth.

Methods for Analyzing Cultivation Experiments

Optical density (OD) at λ=600 nm was determined using aspectrophotometer by pipetting a well mixed broth sample into anappropriate cuvette (CS500 VWR International, Germany). If the biomassconcentration of the sample exceeded the linear absorption range of thespectrophotometer (typically OD values from 0.000 to 0.600), the samplewas diluted with 0.9% NaCl solution to yield values in the linear range.

Metabolites and products in medium were analyzed and quantified using aGC method and an ZB-WAXplus column (30 m×0.25 mm ID, 0.25 μm film) fromPhenomenex (Torrance, Calif.). A helium carrier gas was used at aconstant flow rate of 2.3 mL/min; an injector split of 1:20 at 250° C.;an oven temperature of 70° C. for 1 min, followed by 70° C. to 160° C.at 10° C./min, and 160° C. to 240° C. at 30° C./min. Flame IonizationDetection (FID) was used at 260° C. with 40 mL/min helium makeup gas.Culture broth samples were filtered through 0.2 μm spin filters beforeinjection. 0.5 μl injection volumes were used. Calibrated standardcurves were generated for isobutanol.

Glucose and fermentation by-product analysis were carried out by highperformance liquid chromatography (HPLC) using methods known in the art.The HPLC method utilized a Shodex SH-1011 column with a Shodex SH-Gguard column (both available from Waters Corporation, Milford, Mass.),with refractive index (RI) detection. Chromatographic separation wasachieved using 0.01 N H₂SO₄ as the mobile phase with a flow rate of 0.5mL/min and a column temperature of 50° C. Isobutanol retention time was47.6 minutes.

Isobutanol concentration in the culture supernatant was determined bythe HPLC method. Isobutanol concentration in the oleyl alcohol phase wasdetermined by the GC method. Isobutanol concentration in off-gas sampleswas determined by mass-spec as mentioned above.

Results

The measured values of optical density (OD), isobutanol production rate(R), produced isobutanol per liter of culture broth (isobutanol titer,T), and isobutanol yield per consumed glucose (Y) at about 46 hours offermentation time are presented in Table 5. The PNY2205 strain comparedto PNY1504 and PNY1506 strains grow to higher cell density and resultedin higher titer and rate but similar yield.

TABLE 5 Optical density, isobutanol production rate, titer and yield inPNY1504, PNY1506 and PNY2205 strains R T Y Strain OD (g/L/h) (g/L) (g/g)PNY1504 26.5 0.45 20.7 0.27 PNY1506 27.2 0.55 25.2 0.28 PNY2205 34.60.84 39.7 0.27

Example 7 Comparing the Performance of Strains PNY1504 and PNY2205 Underthe Same Reactive Liquid Extraction Conditions Stock Solutions Used

Pre-Seed Media

The following reagents were mixed with gentle agitation at roomtemperature: 6.7 g of Yeast Nitrogen Base without amino acids (Difco0919-15-3); 2.8 g of Yeast Synthetic Drop-out Medium Supplement WithoutHistidine, Leucine, Tryptophan and Uracil (Sigma Y2001); 20 mL of 1%(w/v) L-Leucine; 4 mL of 1% (w/v) L-Tryptophan; 3 g of ethanol; 3 g ofglucose and enough water to make a total of 1 L of solution.

Seed Flask Media

The following reagents were mixed with gentle agitation at roomtemperature: 6.7 g of Yeast Nitrogen Base without amino acids (Difco0919-15-3); 2.8 g of Yeast Synthetic Drop-out Medium Supplement WithoutHistidine, Leucine, Tryptophan and Uracil (Sigma Y2001); 20 mL of 1%(w/v) L-Leucine; 4 mL of 1% (w/v) L-Tryptophan; 3 g of ethanol; 30 g ofglucose; 38 g of MES buffer (Sigma-Aldrich YXXX) and enough water tomake a total of 1 L of solution. After mixing, the solution was filtersterilized.

Ergosterol Solution

A solution of 0.2 g of Ergosterol, 10 mL of 200 proof ethanol and 10 mlof Tween 80 was mixed and heated to 70° C. for 10 minutes.

Distillase Stock Solution

A solution of 0.9 mL of Distillase L 400 and 49.1 mL of filtersterilized tap water was mixed.

Lipolase 100 L Stock Solution

A solution of 2.12 mL of Lipolase 100 L (Sigma Aldrich L0777) and 40 gof phosphate buffer solution at pH 6.8 were mixed and filter sterilized.

Vitamin Stock Solution

5 g of nicotinic acid and 1 g of thiamine were mixed in 500 mL of filtersterilized Deionized water.

Corn Mash

Corn mash was added to a 30 L liquefaction tank. Next, 16910 g of tapwater was added to the 30 L liquefaction tank with agitation at 120 rpm.The tank was outfitted with a dual-blade pitched-blade turbine withD_(B/DT)=˜0.25. Next, 14091 g of ground corn (ground in a Hammer Millwith a 1 micron screen) was added, and the mash was heated to 55° C. andheld there for 30 minutes. The pH was adjusted to 5.8 by adding 5.4 g of17% NaOH solution in water. An alpha-amylase enzyme solution wasprepared by mixing 1986 g of tap water and 19.5 g of Spezyme Fred L fromGenencor and sterile filtered the resulting solution through a 0.2micron filter. 2004 g of this solution was added to the 30 Lliquefaction tank and held at 55° C. for an additional 60 minutes. Thesolution was then heated to 95° C. and held at that temperature for 120minutes. The solution was cooled to 30° C. before using in fermentation.

PNY1504 Process

Pre-Seed Growth

30 mL of Pre-Seed Media was added to a 250 mL baffled, vented shakeflask. Next, 2 Frozen Seed Vials of Strain PNY1504, ca. 1.5 mL of totalvolume, were added to the same flask. The culture was then incubated for24 hours at 30° C. at 250 rpm on an incubator shaker.

Seed Flask Stage 1

15 mL of the pre-seed culture was added to 300 mL of the Seed Flaskmedia in a 2 L baffled, vented shake flask. The flask was incubated for24 hours at 30° C. and 250 rpm on an incubator shaker.

Seed Flask Stage 2

30 mL of yeast extract peptone and 300 mL of sterile oleyl alcohol werethen added to the flask. The flask was incubated for 24 hours at 30° C.at 250 rpm on an incubator shaker.

1 L Production Fermentor

A 1 L fermentor with water covering the probes was sterilized for 30 minat 121° C. The water was drained and 520 mL of sterile corn mash mediawas added. Next, the following aseptic additions to the corn mash weremade in the fermentor: 3.8 mL of ethanol, 0.6 mL of 1% ergosterolsolution, 6 mL of nicotinic acid/thiamine solution and 4.8 mL ofLiplolase 100 L stock solution. Next, 60 mL of the aqueous phase of SeedFlask Stage 2 was added, followed by 2 mL of the Distillase stocksolution. Directly thereafter, 96 mL of corn oil fatty acid was added.After 12 hours, 2 mL of the Distillase Stock solution was added. At 24hours post inoculation, another 2 mL of Distillase Stock solution wasadded. The solution was then incubated at pH 5.2, temperature 30° C. anda pO2 (partial pressure of dissolved oxygen) setpoint of 3%. Airflow wasset at 0.2 slpm (standard liters per minute) and the pO2 was controlledvia agitation. pH was controlled with 20% w/v KOH solution and no acidwas required throughout the fermentation. Samples were taken andanalyzed over the course of the fermentation.

PNY2205 Process

Pre-Seed Growth

30 mL of Pre-Seed Media was added to a 250 mL baffled, vented shakeflask. Next, 2 Frozen Seed Vials of Strain PNY2205, ca. 1.5 mL of totalvolume, were added to the same flask. We then held the flask for 24hours at 30° C. at 250 rpm on an incubator shaker.

Seed Flask Stage 1

300 mL of the Seed Flask media was added to a 2 L baffled, vented shakeflask. 15 mL of pre-seed culture was added flask and incubated for 24hours at 30° C. and 250 rpm on an incubator shaker.

Seed Flask Stage 2

30 mL of yeast extract peptone and 300 mL of sterile oleyl alcohol wasadded to the flask and the flask was incubated for 24 hours at 30° C.and 250 rpm on an incubator shaker.

1 L Production Fermentor

A 1 L fermentor with water covering the probes was sterilized for 30 minat 121° C. The water was drained and 520 mL of sterile corn mash mediawas added. Next, the following aseptic additions were made to the cornmash in the fermentor: 3.8 mL of ethanol, 0.6 mL of 1% ergosterolsolution, 6 mL of nicotinic acid/thiamine solution and 4.8 mL ofLiplolase 100 L stock solution. Next, 60 mL of the aqueous phase of SeedFlask Stage 2 was added, followed by 2 mL of the Distillase stocksolution. Directly thereafter, 96 mL of corn oil fatty acid was added.12 hours post inoculation, 2 mL of the Distillase Stock solution wasadded. At 24 hours post inoculation, 2 mL of Distillase Stock solutionwas also added. The solution was incubated at pH 5.2, temperature 30° C.and the pO2 setpoint of 3%. Airflow was set at 0.2 slpm and the pO2 wascontrolled via agitation. pH was controlled with 20% w/v KOH solutionand no acid was required throughout the fermentation. Samples were takenand analyzed over the course of the fermentation.

Methods for Analyzing Cultivation Experiments

Optical density (OD) of the resulting cultures was measured at X=600 nmusing a spectrophotometer. First, a well mixed broth sample was pipettedinto an appropriate cuvette. When the biomass concentration of thesample exceeded the linear absorption range of the spectrophotometer(typically OD values from 0.000 to 0.600), the sample was diluted with0.9% NaCl solution to yield values in the linear range. Dry weight ofthe cell suspension was determined by centrifuging 5 mL of cell broth ina pre-weighed centrifuge tube, followed by washing with distilled water,drying to constant weight at 80° C. in an oven and determining theweight difference.

Metabolites and products in medium were analyzed and quantified by a GCmethod utilizing a ZB-WAXplus column (30 m×0.25 mm ID, 0.25 μm film)from Phenomenex (Torrance, Calif.). The carrier gas was helium at aconstant flow rate of 2.3 mL/min; injector split was 1:20 at 250° C.;oven temperature is 70° C. for 1 min, 70° C. to 160° C. at 10° C./min,and 160° C. to 240° C. at 30° C./min. FID detection was used at 260° C.with 40 ml/min helium makeup gas. Culture broth samples were filteredthrough 0.2 μm spin filters before injection. A calibrated standardcurve for isobutanol (w-methyl-1-propanol) was used.

Glucose analysis was carried out by YSI (YSI 2700 Select biochemistryanalyzer that uses enzyme electrode technology to generate rapidmeasurement of glucose concentration.

Results

Isobutanol production rate, isobutanol per liter of culture broth(effective titer), and isobutanol yield per consumed glucose arepresented in Table 6. The PNY2205 strain compared to PNY1504 strainsresulted in higher production rate and titer but similar yield.

TABLE 6 Optical density and isobutanol production of PNY2205 compared toPNY1504 52-56 hr result PNY1504 PNY2205 rate, g/L-h 0.50 0.64 effectivetiter 26.3 35.5 (g/L) g/g glu yield 0.27 0.27

Example 8 Comparing the Performance of Strains PNY1504 and PNY2205 Underthe Same Reactive Liquid Extraction Conditions Stock Solutions Used

Pre-Seed Media

The following reagents were mixed with gentle agitation at roomtemperature: 6.7 g of Yeast Nitrogen Base without amino acids (Difco0919-15-3); 2.8 g of Yeast Synthetic Drop-out Medium Supplement WithoutHistidine, Leucine, Tryptophan and Uracil (Sigma Y2001); 20 mL of 1%(w/v) L-Leucine; 4 mL of 1% (w/v) L-Tryptophan; 3 g of ethanol; 3 g ofglucose and enough water to make a total of 1 L of solution.

Seed Flask Media

The following reagents were mixed with gentle agitation at roomtemperature: 6.7 g of Yeast Nitrogen Base without amino acids (Difco0919-15-3); 2.8 g of Yeast Synthetic Drop-out Medium Supplement WithoutHistidine, Leucine, Tryptophan and Uracil (Sigma Y2001); 20 mL of 1%(w/v) L-Leucine; 4 mL of 1% (w/v) L-Tryptophan; 3 g of ethanol; 30 g ofglucose; 38 g of MES buffer (Sigma-Aldrich YXXX) and enough water tomake a total of 1 L of solution. After mixing, the solution was filtersterilized.

Ergosterol Solution

A solution of 0.2 g of Ergosterol, 10 mL of 200 proof ethanol and 10 mLof Tween 80 was mixed and heated to 70° C. for 10 minutes.

Distillase Stock Solution

A solution of 0.9 mL of Distillase L 400 and 49.1 mL of filtersterilized tap water was mixed.

Lipolase 100 L Stock Solution

A solution of 2.12 mL of Lipolase 100 L (Sigma Aldrich L0777) and 40 gof phosphate buffer solution at pH 6.8 was mixed and filter sterilized.

Vitamin Stock Solution

A solution of 5 g of nicotinic acid and 1 g of thiamine in was mixed in500 mL of filter sterilized Deionized water.

Corn Mash

Corn mash was added to a 30 L liquefaction tank. Next, 16910 g of tapwater was added to the 30 L liquefaction tank with agitation at 120 rpm.The tank was outfitted with a dual-blade pitched-blade turbine withD_(B/DT)=˜0.25. Next, 14091 g of ground corn (ground in a Hammer Millwith a 1 micron screen) was added and the mash heated to 55° C. andincubated for 30 minutes. The pH was adjusted to 5.8 by adding 5.4 g of17% NaOH solution in water. An alpha-amylase enzyme solution wasprepared by mixing 1986 g of tap water and 19.5 g of Spezyme Fred L fromGenencor and sterile filtering through a 0.2 micron filter. 2004 g ofthis solution was added to the 30 L liquefaction tank and incubated at55° C. for an additional 60 minutes. Then, the solution was heated to95° C. and held there for 120 minutes. The solution was then cooled to30° C. before using in fermentation.

PNY1504 Process

Pre-Seed Growth

30 mL of Pre-Seed Media was added to a 250 mL baffled, vented shakeflask. Next, 2 Frozen Seed Vials of Strain PNY1504, ca. 1.5 ml of totalvolume, were added to the same flask. The culture was incubated for 24hours at 30° C. at 250 rpm on an incubator shaker.

Seed Flask Stage 1

300 mL of the Seed Flask media was added to a 2 L baffled, vented shakeflask. 15 mL of pre-seed was then transferred to flask. The flask wasthen incubated for 24 hours at 30° C. and 250 rpm on an incubatorshaker.

Seed Flask Stage 2

30 mL of yeast extract peptone and 300 mL of sterile oleyl alcohol wereadded to the flask and the flask incubated for 24 hours at 30° C. at 250rpm on an incubator shaker.

1 L Production Fermentor

A 1 L fermentor with water covering the probes was sterilized for 30 minat 121° C. The water was drained and 520 mL of sterile corn mash mediawas added. Next, the following aseptic additions were made to the cornmash in the fermentor: 3.8 mL of ethanol, 0.6 mL of 1% ergosterolsolution, 6 mL of nicotinic acid/thiamine solution and 4.8 mL ofLiplolase 100 L stock solution. Next, 60 mL of the aqueous phase of SeedFlask Stage 2 was added, followed by 2 mL of the Distillase stocksolution. Directly thereafter, 141 mL of corn oil fatty acid was added.At 12 hours post inoculation, 2 mL of the Distillase Stock solution wasadded. At 24 hours post inoculation, 2 mL of Distillase Stock solutionwas also added. The solution was then incubated at pH 5.2, temperature30° C. and pO2 setpoint of 3%. Airflow was set at 0.2 slpm and pO2 wascontrolled via agitation. pH was controlled with 20% w/v KOH solutionand no acid was required throughout the fermentation. Samples were takenand analyzed over the course of the fermentation.

PNY2205 Process

Pre-Seed Growth

30 mL of Pre-Seed Media was added to a 250 mL baffled, vented shakeflask. Next, 2 Frozen Seed Vials of Strain PNY2205, ca. 1.5 ml of totalvolume, were added to the same flask. The flask was then incubated for24 hours at 30° C. at 250 rpm on an incubator shaker.

Seed Flask Stage 1

300 mL of the Seed Flask media was added to a 2 L baffled, vented shakeflask. 15 mL of the pre-seed growth was then added to the flask andincubated for 24 hours at 30° C. and 250 rpm on an incubator shaker.

Seed Flask Stage 2

30 mL of yeast extract peptone and 300 mL of sterile oleyl alcohol wereadded to the flask and incubated for 24 hours at 30° C. and 250 rpm onan incubator shaker.

1 L Production Fermentor

A 1 L fermentor with water covering the probes was sterilized for 30 minat 121° C. The water was drained and 520 mL of sterile corn mash mediawas added. Next, the following aseptic additions were made to the cornmash in the fermentor: 3.8 mL of ethanol, 0.6 mL of 1% ergosterolsolution, 6 mL of nicotinic acid/thiamine solution and 4.8 mL ofLiplolase 100 L stock solution. Next, 60 mL of the aqueous phase of SeedFlask Stage 2 was added followed by 2 mL of the Distillase stocksolution. Directly thereafter, 96 mL of corn oil fatty acid was added.At 12 hours post inoculation, 2 mL of the Distillase Stock solution wasadded. At 24 hours post inoculation, 2 mL of Distillase Stock solutionwas also added and the solution was incubated at pH 5.2, temperature 30°C. and pO2 setpoint of 3%. Airflow was set at 0.2 slpm and pO2 wascontrolled via agitation. pH was controlled with 20% w/v KOH solutionand no acid was required throughout the fermentation. Samples were takenand analyzed over the course of the fermentation.

Results

Isobutanol production rate, isobutanol per liter of culture broth(effective titer), and isobutanol yield per consumed glucose arepresented in Table 7. The PNY2205 strain compared to PNY1504 strainsresulted in higher production rate and titer but similar yield.

TABLE 7 Optical density and isobutanol production of PNY2205 compared toPNY1504 52-56 hr result PNY1504 PNY2205 rate, g/l-h 0.48 0.54 effectivetiter (g/l) 25.2 30.1 g/g glu yield 0.27 0.27

Example 9 Comparing the Performance of Strains PNY1504 and PNY2205 Underthe Same Reactive Liquid Extraction Conditions Stock Solutions Used

Pre-Seed Media

The following reagents were mixed with gentle agitation at roomtemperature: 6.7 g of Yeast Nitrogen Base without amino acids (Difco0919-15-3); 2.8 g of Yeast Synthetic Drop-out Medium Supplement WithoutHistidine, Leucine, Tryptophan and Uracil (Sigma Y2001); 20 mL of 1%(w/v) L-Leucine; 4 mL of 1% (w/v) L-Tryptophan; 3 g of ethanol; 3 g ofglucose and enough water to make a total of 1 L of solution.

Seed Flask Media

The following reagents were mixed with gentle agitation at roomtemperature: 6.7 g of Yeast Nitrogen Base without amino acids (Difco0919-15-3); 2.8 g of Yeast Synthetic Drop-out Medium Supplement WithoutHistidine, Leucine, Tryptophan and Uracil (Sigma Y2001); 20 mL of 1%(w/v) L-Leucine; 4 mL of 1% (w/v) L-Tryptophan; 3 g of ethanol; 30 g ofglucose; 38 g of MES buffer (Sigma-Aldrich YXXX) and enough water tomake a total of 1 L of solution. After mixing, the solution was filtersterilized.

Ergosterol Solution

A solution of 0.2 g of Ergosterol, 10 mL of 200 proof ethanol and 10 mLof Tween 80 was mixed and heated to 70° C. for 10 minutes.

Distillase Stock Solution

A solution of 0.9 mL of Distillase L 400 and 49.1 mL of filtersterilized tap water was mixed.

Lipolase 100 L Stock Solution

A solution of 2.12 mL of Lipolase 100 L (Sigma Aldrich L0777) and 40 gof phosphate buffer solution at pH 6.8 was mixed and filter sterilized.

Vitamin Stock Solution

A solution of 5 g of nicotinic acid and 1 g of thiamine in was mixed in500 mL of filter sterilized Deionized water.

Corn Mash

Corn mash was added to a 30 L liquefaction tank. Next, 16910 g of tapwater was added to the 30 L liquefaction tank with agitation at 120 rpm.The tank was outfitted with a dual-blade pitched-blade turbine withD_(B/DT)=˜0.25. Next, 14091 g of ground corn (ground in a Hammer Millwith a 1 micron screen) was added and the mash heated to 55° C. andincubated for 30 minutes. The pH was adjusted to 5.8 by adding 5.4 g of17% NaOH solution in water. An alpha-amylase enzyme solution wasprepared by mixing 1986 g of tap water and 19.5 g of Spezyme Fred L fromGenencor and sterile filtering through a 0.2 micron filter. 2004 g ofthis solution was added to the 30 L liquefaction tank and incubated at55° C. for an additional 60 minutes. Then, the solution was heated to95° C. and held there for 120 minutes. The solution was then cooled to30° C. before using in fermentation.

PNY1504 Process

Pre-Seed Growth

30 mL of Pre-Seed Media was added to a 250 mL baffled, vented shakeflask. Next, 2 Frozen Seed Vials of Strain PNY1504, ca. 1.5 ml of totalvolume, were added to the same flask. The culture was incubated for 24hours at 30° C. at 250 rpm on an incubator shaker.

Seed Flask Stage 1

300 mL of the Seed Flask media was added to a 2 L baffled, vented shakeflask. 15 mL of pre-seed was then transferred to flask. The flask wasthen incubated for 24 hours at 30° C. and 250 rpm on an incubatorshaker.

Seed Flask Stage 2

30 mL of yeast extract peptone and 300 mL of sterile oleyl alcohol wereadded to the flask and the flask incubated for 24 hours at 30° C. at 250rpm on an incubator shaker.

1 L Production Fermentor

A 1 L fermentor with water covering the probes was sterilized for 30 minat 121° C. The water was drained and 520 mL of sterile corn mash mediawas added. Next, the following aseptic additions were made to the cornmash in the fermentor: 3.8 mL of ethanol, 0.6 mL of 1% ergosterolsolution, 6 mL of nicotinic acid/thiamine solution and 4.8 mL ofLiplolase 100 L stock solution. Next, 60 mL of the aqueous phase of SeedFlask Stage 2 was added, followed by 2 mL of the Distillase stocksolution. Directly thereafter, 141 mL of corn oil fatty acid was added.At 12 hours post inoculation, 2 mL of the Distillase Stock solution wasadded. At 24 hours post inoculation, 2 mL of Distillase Stock solutionwas also added. The solution was then incubated at pH 5.2, temperature30° C. and pO2 setpoint of 3%. Airflow was set at 0.2 slpm and pO2 wascontrolled via agitation. pH was controlled with 20% w/v KOH solutionand no acid was required throughout the fermentation. Samples were takenand analyzed over the course of the fermentation.

PNY2205 Process

Pre-Seed Growth

30 mL of Pre-Seed Media was added to a 250 mL baffled, vented shakeflask. Next, 2 Frozen Seed Vials of Strain PNY2205, ca. 1.5 ml of totalvolume, were added to the same flask. The flask was then incubated for24 hours at 30° C. at 250 rpm on an incubator shaker.

Seed Flask Stage 1

300 mL of the Seed Flask media was added to a 2 L baffled, vented shakeflask. 15 mL of the pre-seed growth was then added to the flask andincubated for 24 hours at 30° C. and 250 rpm on an incubator shaker.

Seed Flask Stage 2

30 mL of yeast extract peptone and 300 mL of sterile oleyl alcohol wereadded to the flask and incubated for 24 hours at 30° C. and 250 rpm onan incubator shaker.

1 L Production Fermentor

A 1 L fermentor with water covering the probes was sterilized for 30 minat 121° C. The water was drained and 520 mL of sterile corn mash mediawas added. Next, the following aseptic additions were made to the cornmash in the fermentor: 3.8 mL of ethanol, 0.6 mL of 1% ergosterolsolution, 6 mL of nicotinic acid/thiamine solution and 4.8 mL ofLiplolase 100 L stock solution. Next, 60 mL of the aqueous phase of SeedFlask Stage 2 was added followed by 2 mL of the Distillase stocksolution. Directly thereafter, 96 mL of corn oil fatty acid was added.At 12 hours post inoculation, 2 mL of the Distillase Stock solution wasadded. At 24 hours post inoculation, 2 mL of Distillase Stock solutionwas also added and the solution was incubated at pH 5.2, temperature 30°C. and pO2 setpoint of 3%. Airflow was set at 0.2 slpm and pO2 wascontrolled via agitation. pH was controlled with 20% w/v KOH solutionand no acid was required throughout the fermentation. Samples were takenand analyzed over the course of the fermentation.

Results

Isobutanol production rate, isobutanol per liter of culture broth(effective titer), and isobutanol yield per consumed glucose arepresented in Table 8. The PNY2205 strain compared to PNY1504 strainsresulted in higher production rate and titer but similar yield.

TABLE 8 Isobutanol production of PNY2205 compared to PNY1504 52-56 hrresult PNY1504 PNY2205 rate, g/l-h 0.51 0.58 effective titer (g/l) 26.732.6 g/g glu yield 0.27 0.27

Example 10 Construction of S. cerevisiae Strain PNY2211

PNY2211 was constructed in several steps from S. cerevisiae strainPNY1507 (Example 2) as described in the following paragraphs. First, thestrain was modified to contain a phosphoketolase gene. Construction ofphosphoketolase gene cassettes and integration strains was previouslydescribed in U.S. Appl. No. 61/356,379, filed Jun. 18, 2010. Next, anacetolactate synthase gene (alsS) was added to the strain, using anintegration vector described in Example 3. Finally, homologousrecombination was used to remove the phosphoketolase gene andintegration vector sequences, resulting in a scarless insertion of alsSin the intergenic region between pdc1Δ::ilvD (a previously describeddeletion/insertion of the PDC1 ORF, U.S. Appl. No. 61/356,379, filedJun. 18, 2010; see Example 1 herein) and the native TRX1 gene ofchromosome XII. The resulting genotype of PNY2211 is MATa ura3Δ::loxPhis3Δ pdc6Δ pdc1Δ::P[PDC1]-DHAD|ilvD_Sm-PDC1t-P[FBA1]-ALS|alsS_Bs-CYC1tpdc5Δ::P[PDC5]-ADH|sadB_Ax-PDC5t gpd2Δ::loxP fra2Δadh1Δ::UAS(PGK1)P[FBA1]-kivD_Ll(y)-ADH1t.

A phosphoketolase gene cassette was introduced into PNY1507 byhomologous recombination. The integration construct was generated asfollows. The plasmid pRS423::CUP1-alsS+FBA-budA (described in U.S. Pub.No. 2009/0305363 A1) was digested with NotI and XmaI to remove the 1.8kb FBA-budA sequence, and the vector was religated after treatment withKlenow fragment. Next, the CUP1 promoter was replaced with a TEF1promoter variant (M4 variant described by Nevoigt et al. Appl. Environ.Microbiol. 2006. 72(8): 5266-5273) via DNA synthesis and vectorconstruction service from DNA2.0 (Menlo Park, Calif.). The resultingplasmid, pRS423::TEF(M4)-alsS was cut with StuI and MluI (removes 1.6 kbportion containing part of the alsS gene and CYC1 terminator), combinedwith the 4 kb PCR product generated from pRS426::GPD-xpk1+ADH-eutD(described in U.S. Appl. No. 61/356,379, filed Jun. 18, 2010; SEQ ID NO:246 herein) with primers N1176 (SEQ ID NO:207) and N1177 (SEQ ID NO:208)and an 0.8 kb PCR product DNA generated from yeast genomic DNA (ENO1promoter region) with primers N822 (SEQ ID NO:209) and N1178 (SEQ IDNO:210) and transformed into S. cerevisiae strain BY4741 (ATCC 201388;gap repair cloning methodology, see Ma and Botstein). Transformants wereobtained by plating cells on synthetic complete medium withouthistidine. Proper assembly of the expected plasmid(pRS423::TEF(M4)-xpk1+ENO1-eutD, SEQ ID NO:211) was confirmed by PCR(primers N821 (SEQ ID NO:212) and N1115 (SEQ ID NO:213)) and byrestriction digest (BglI). Two clones were subsequently sequenced. The3.1 kb TEF(M4)-xpk1 gene was isolated by digestion with SacI and NotIand cloned into the pUC19-URA3::ilvD-TRX1 vector (described in U.S.Appl. No. 61/356,379, filed Jun. 18, 2010 SEQ ID NO: 243, herein) CloneA, cut with AflII). Cloning fragments were treated with Klenow fragmentto generate blunt ends for ligation. Ligation reactions were transformedinto E. coli Stbl3 cells, selecting for ampicillin resistance. Insertionof TEF(M4)-xpk1 was confirmed by PCR (primers N1110 (SEQ ID NO:214) andN1114 (SEQ ID NO:215)). The vector was linearized with AflII and treatedwith Klenow fragment. The 1.8 kb KpnI-HincII geneticin resistancecassette (described in U.S. Appl. No. 61/356,379, filed Jun. 18, 2010;SEQ ID NO: 245 herein), was cloned by ligation after Klenow fragmenttreatment. Ligation reactions were transformed into E. coli Stbl3 cells,selecting for ampicillin resistance. Insertion of the geneticin cassettewas confirmed by PCR (primers N160SeqF5 (SEQ ID NO:216) and BK468 (SEQID NO:217)). The plasmid sequence is provided as SEQ ID NO:218(pUC19-URA3::pdc1::TEF(M4)-xpk1::kan).

The resulting integration cassette (pdc1::TEF(M4)-xpk1::KanMX::TRX1) wasisolated (AscI and NaeI digestion generated a 5.3 kb band that was gelpurified) and transformed into PNY1507 (Example 2) using the ZymoResearch Frozen-EZ Yeast Transformation Kit (Cat. No. T2001).Transformants were selected by plating on YPE plus 50 μg/ml G418.Integration at the expected locus was confirmed by PCR (primers N886(SEQ ID NO:219) and N1214 (SEQ ID NO:220)). Next, plasmidpRS423::GAL1p-Cre, encoding Cre recombinase, was used to remove theloxP-flanked KanMX cassette (vector and methods described herein).Proper removal of the cassette was confirmed by PCR (primers oBP512 (SEQID NO:221) and N160SeqF5 (SEQ ID NO:222)). Finally, the alsS integrationplasmid described herein (pUC19-kan::pdc1::FBA-alsS::TRX1, clone A) wastransformed into this strain using the included geneticin selectionmarker. Two integrants were tested for acetolactate synthase activity bytransformation with plasmids pYZ090ΔalsS and pBP915 (plasmids describedherein, transformed using Protocol #2 in “Methods in Yeast Genetics”2005. Amberg, Burke and Strathern) and evaluation of growth andisobutanol production in glucose-containing media (methods for growthand isobutanol measurement are described herein and U.S. Appl. No.60/730,290, filed Oct. 26, 2005 and U.S. Pub. No. 2007/0092957 A1). Oneof the two clones was positive and was named PNY2218. An isolate ofPNY2218 containing the plasmids pYZ090ΔalsS and pBP915 was designatedPNY2209.

PNY2218 was treated with Cre recombinase and resulting clones werescreened for loss of the xpk1 gene and pUC19 integration vectorsequences by PCR (primers N886 (SEQ ID NO:219) and N160SeqR5 (SEQ IDNO:222)). This leaves only the alsS gene integrated in the pdc1-TRX1intergenic region after recombination the DNA upstream of xpk1 and thehomologous DNA introduced during insertion of the integration vector (a“scarless” insertion since vector, marker gene and loxP sequences arelost, FIG. 6). Although this recombination could have occurred at anypoint, the vector integration appeared to be stable even withoutgeneticin selection and the recombination event was only observed afterintroduction of the Cre recombinase. One clone was designated PNY2211.

Example 11 Comparing the Performance of Strains PNY2205 and PNY2211Under the Same Reactive Liquid Extraction Conditions

Isolates with the scarless integration (in particular, two clones “B”and “M”) were transformed with pYZ090ΔalsS and pBP915 in order tocompare isobutanol production with PNY2205. Integrants were selected onsynthetic complete medium (minus histidine and uracil) containing 1%ethanol as the carbon source. Integrants were patched to the samemedium, and patched cells were patched again to plates containing 2%glucose plus 0.05% ethanol as carbon sources. After two days, patcheswere used to inoculate liquid medium (10 mL synthetic complete, minushistidine and uracil, with 2% glucose and 0.05% ethanol in 125 mL ventedflasks). After overnight incubation (30° C., 250 rpm) cultures werediluted back to OD 0.2 (20 mL medium in 125 mL tightly capped flasks).After 48 hours, samples were taken to determine isobutanol production.The new strain backgrounds supported similar isobutanol production toPNY2205. Clone M was selected for further engineering was named PNY2211.Clone M7 transformed with plasmids pYZ090DalsS and pBP915 was designatedPNY2213.

The production of isobutanol per liter of culture broth (effective titerin g/L) of strains PNY2205, Clone B and Clone M is presented in Table 9.Clone B and M strains had a similar isobutanol titer compared toPNY2205.

TABLE 9 Isobutanol production of PNY2205 compared to PNY2211 StrainsIsobutanol titer (g/L) PNY2205 4.0 Clone B strains (n = 3) 3.8 +/− 0.5Clone M strains (n = 3) (PNY2213) 4.0 +/− 0.5

1-47. (canceled)
 48. A recombinant host cell comprising: (a) apolynucleotide encoding a polypeptide which catalyzes the substrate toproduct conversion of pyruvate to acetolactate wherein the polypeptideis an acetolactate synthase from Bacillus subtilis, Klebsiellapneumonia, Lactococcus lactis, Staphylococcus aureus, Listeriamonocytogenes, Streptococcus mutans, Streptococcus thermophiles, Vibrioangustum, or Bacillus cereus. (b) a polynucleotide encoding apolypeptide which catalyzes the substrate to product conversion ofacetolactate to 2,3-dihydroxyisovalerate wherein the polypeptide is aketol-acid reductoisomerase from Lactococcus lactis, Vibrio cholera,Pseudomonas aeruginosa, Pseudomonas fluorescens, or Anaerostipes caccae.(c) a polynucleotide encoding a polypeptide which catalyzes thesubstrate to product conversion of 2,3-dihydroxyisovalerate toα-ketoisovalerate wherein the polypeptide is a dihydroxyacid dehydratasefrom Escherichia coli, Bacillus subtilis, Methanococcus maripaludis, orStreptococcus mutans; and (d) a polynucleotide encoding a polypeptidewhich catalyzes the substrate to product conversion of α-ketoisovalerateto isobutyraldehyde wherein the polypeptide is a branched-chain α-ketoacid decarboxylase from Listeria grayi, Lactococcus lactis, orMacrococcus caseolyticus.
 49. The recombinant host cell of claim 48further comprising a polynucleotide encoding a polypeptide whichcatalyzes the substrate to product conversion isobutyraldehyde toisobutanol wherein the polypeptide is an alcohol dehydrogenase fromAchromobacter xylosoxidans or Beijerinkia indica.
 50. The recombinanthost cell of claim 48, wherein the acetolactate synthase has at least95% identity to an amino acid sequence selected from SEQ ID NOs: 2, 4,6, 8, 10, 12, 14, 16, or
 18. 51. The recombinant host cell of claim 48,wherein the ketol-acid reductoisomerase has at least 95% identity to anamino acid sequence selected from SEQ ID NOs: 40, 42, 44, 224, or 225.52. The recombinant host cell of claim 48, wherein the dihydroxyaciddehydratase has at least 95% identity to amino acid sequence of SEQ IDNO:
 89. 53. The recombinant host cell of claim 48, wherein thebranched-chain α-keto acid decarboxylase has at least 95% identity to anamino acid sequence selected from SEQ ID NOs: 48, 247, or
 248. 54. Therecombinant host cell of claim 49, wherein the alcohol dehydrogenase hasat least 95% identity to an amino acid sequence selected from SEQ IDNOs: 36 or
 237. 55. The recombinant host cell of claim 48, whereinexpression of pyruvate decarboxylase in the recombinant host cell isdecreased or substantially eliminated.
 56. The recombinant host cell ofclaim 48, wherein the recombinant host cell comprises a deletion,mutation, and/or substitution in an endogenous polynucleotide encoding apolypeptide having pyruvate decarboxylase activity.
 57. The recombinanthost cell of claim 48, wherein expression of glycerol-3-phosphatedehydrogenase in the recombinant host cell is decreased or substantiallyeliminated.
 58. The recombinant host cell of claim 48, whereinexpression of Fra2 in the recombinant host cell is decreased orsubstantially eliminated.
 59. The recombinant host cell of claim 48,wherein expression of pyruvate decarboxylase, glycerol-3-phosphatedehydrogenase, and Fra2 in the recombinant host cell is decreased orsubstantially eliminated.
 60. The recombinant host cell of claim 48,wherein the recombinant host cell is selected from bacterium,cyanobacterium, filamentous fungus, or yeast.
 61. The recombinant hostcell of claim 48, wherein the recombinant host cell is selected from thegroup consisting of Clostridium, Zymomonas, Escherichia, Salmonella,Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus,Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Pichia, Candida, Hansenula, Issatchenkia, Kluyveromyces,and Saccharomyces.
 62. The recombinant host cell of claim 48, whereinthe recombinant host cell is selected from the group consisting ofSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Kluyveromyces thermotolerans, Candida glabrata, Candidaalbicans, Pichia stipitis, and Yarrowia lipolytica.
 63. A methodcomprising (a) providing the recombinant host cell of claim 48; and (b)contacting the recombinant host cell with a fermentable carbon substrateunder conditions whereby a product is produced.
 64. The method of claim63, wherein the product is isobutanol.